| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Academic Affairs, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90048
 |
Abstract |
|---|
 Top Abstract I. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
|
I. Introduction |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine of the interleukin-6 (IL-6) cytokine family, sharing the common gp130 receptor subunit together with IL-6, interleukin-11 (IL-11), oncostatin (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin (CT-1). The leukemia inhibitory factor receptor (LIFR) is a class I cytokine receptor, belonging to the hematopoietic cytokine receptor superfamily. In addition to classical hematopoietic effects, LIF affects various endocrine tissues and cell types, including proliferation of primordial germ cells, maintenance of pluripotent embryonal stem cells, endometrial decidualization and blastocyst implantation, hypothalamus-pituitary-adrenal (HPA) axis activation and pituitary development, osteoblast and osteoclast function, adipocyte lipid and energy homeostasis, and auto/paracrine growth regulation of endocrine-responsive neoplasms.
The topic of LIF in the endocrine system was reviewed in this journal in 1991 by Kurzrock et al. (1), and several recent reviews have summarized general aspects of LIF action (2, 3, 4, 5). In the past few years, however, significant new knowledge has been gained on both LIF signaling as well as immune-endocrine functions of LIF. Recent studies have highlighted the LIF-induced Jak-STAT (janus kinase-signal transducer and activator of transcription) signaling cascade, and its negative feedback-regulation by suppressor-of cytokine-signaling proteins (SOCS) and protein inhibitors of activated STAT (PIAS). A number of recent animal and human studies have indicated an important immune-endocrine role for LIF in blastocyst implantation and early pregnancy. Recent studies in infertile women suggest a potential link for LIF in unexplained failure of implantation in humans. An important functional role for LIF as a neuroimmune-endocrine modulator in the hypothalamo-pituitary-adrenal axis and in pituitary development has recently been demonstrated. These findings have strong pathophysiological implications on the role of LIF in the HPA axis response to various afferent stimuli including stress and inflammation. There is also increasing evidence favoring a significant role for LIF in bone development and metabolism, energy metabolism, and as an auto/paracrine growth factor in endocrine-responsive tumors, including breast cancer.
The recent unraveling of the LIF-induced Jak-STAT signaling and SOCS-mediated autoregulatory feedback, as well as the immune-endocrine function of LIF in blastocyst implantation and infertility and the neuroimmune-endocrine modulation of HPA axis activity, link LIF to currently important and topical areas of endocrine research. Taking into account the recent enhanced understanding of this ubiquitous cytokine and its various functions, this review therefore focuses on the LIF signaling cascade and its immuno-endocrine functions.
|
II. LIF—Gene Structure and Regulation |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
A. LIF gene and primary structure
To date, the murine (GenBank Accession X06381, M63419 J05435,
X12810 M60289, S73374) (7, 8, 9, 10, 11, 12), human (GenBank Accession
M63420 J05436, X13967) (12, 13, 14, 15, 16, 17, 18), porcine (19), ovine (19), bovine
(GenBank Accession D50337, U63311) (20, 21), rat (22), and mink
(GenBank Accession AF048827) (23) genes for LIF have been cloned.
Southern blot analysis with human and murine probes of the LIF coding region yield a unique hybridization pattern (12), indicating a single gene locus. The murine LIF gene is located on chromosome 11A1 (24, 25), while the human LIF gene is located on chromosome 22q12.1–12.2 (15, 16, 26, 27, 28). In the murine and human genome, the LIF gene is in close proximity to the OSM gene (15, 16, 24, 25, 26, 27, 28, 29).
The length of the murine and human LIF gene is approximately 6.0 kb and 6.3 kb, respectively (12). Northern blot analysis with a specific murine LIF probe detects a single approximately 4.2-kb transcript (12) and a similar transcript size is found for human LIF. The human and murine LIF gene consist of 3 exons and 2 introns (12). Exon 1 encodes the first 6 amino acids of the hydrophobic leader, exon 2 encodes the rest of the hydrophobic leader and the first 53 amino acids of the mature protein, while exon 3 encodes the C-terminal 137 amino acids and an extremely long 3'-untranslated region spanning approximately 3.2 kb (12). The human and murine LIF genes show a high homology of 78–94% in their coding regions (12, 19), while the noncoding regions are much less conserved. At the amino acid level, human and murine LIF show 79% homology (19). Murine glycosylated LIF is a 38- to 67-kDa protein (5, 6), which can be deglycosylated to an approximately 20-kDa protein consisting of 180 amino acids (aa), without losing its biological activity (5, 6). In contrast, N-linked glycosylation of rat LIF at various sites has been shown to differently alter LIF bioactivity in a cell-specific manner (30, 31).
The minimal LIF promoter extends from -103 to +1 (31), and this region is totally conserved between murine, human, ovine, and porcine LIF genes, except for a single insertion (19). The major transcription start site has been located 60–64 bp upstream of the translation initiation codon (12). Further functional elements in the 5'-region of murine LIF are negative regulatory elements between -360 and -249 (31) and in a GC-rich hypomethylated region between the respective first exons of diffusible and matrix-associated LIF (11, 32). Distal positive enhancers, which can overcome the negative elements, are located in the murine LIF gene at -860 to -661 (33) and at -3,200 to -1,200 nucleotides (11). The presence of negative regulatory elements might explain the very low constitutive expression of LIF in most tissues, which, however, can be induced by several cytokines and mitogens.
In addition to the originally described form of murine LIF, designated diffusible LIF (LIF-D), an alternative form, termed matrix-associated LIF (LIF-M), has been described (11, 34). Initially, matrix-associated LIF-M has been considered a murine entity, as it was not found in the human genome (11, 19, 34). However, recent studies demonstrate the existence of human and porcine LIF-M (35, 36). In addition, a truncated LIF version (LIF-T) was found in the murine, human, and porcine genome (35, 36). These findings indicate a complex and conserved organization of the mammalian LIF gene (35, 36). Expression of LIF-D, LIF-M, and LIF-T transcripts differs in a cell-specific manner (35, 36). LIF-M and LIF-T arise by alternate promoter usage and splicing of different exons 1 to exon 2 and 3 (11, 34, 35). The respective DNA sequences encoding exon 1 of LIF-M and LIF-T are located within the first intron of LIF-D (11, 19, 34, 35, 36). LIF-T lacks an in-frame initiation codon in exon 1, and a truncated approximately 17-kDa LIF protein is translated from a transcript, initiated by an in-frame initiation codon in exon 2 (35, 36). LIF-T is expressed intracellularly, but no significant amounts are secreted (35, 36). This is due to protein translation initiated downstream of the secretion signaling sequence (35, 36). Exon 1 of murine LIF-M harbors an in-frame initiation codon, and the resulting N-terminal protein region directs LIF protein secretion to the extracellular matrix (11, 19, 34). Human LIF-M—similar to LIF-T transcripts—lacks an in-frame initiation codon in exon 1 (36) but also does not utilize the in-frame initiation codon in exon 2 (36). As significant amounts of a secreted 20-kDa LIF protein are translated from human LIF-M transcripts (36), an atypical mode of translation initiation using a non-AUG codon has been suggested (36).
B. LIF expression
LIF is expressed and secreted in a variety of tissues and cell
types (for review see Ref. 6). Basal LIF tissue expression is usually
low and often not detectable by Northern blot analysis (6, 37). LIF
gene expression can be induced by several proinflammatory agents,
e.g., lipopolysaccharide (6, 37, 38), IL-1 (6, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49),
IL-17 (48), and tumor necrosis factor-
(TNF-) (6, 39, 40, 41, 42, 45, 47, 49), or inhibited by antiinflammatory agents, e.g.,
glucocorticoids (40, 44, 50, 51), IL-4 (43, 46, 48), and IL-13 (48),
respectively.
C. LIF protein tertiary structure
LIF is a long-chain four--helix bundle cytokine (5, 52, 53, 54, 55, 56, 57, 58, 59, 60).
The four--helix bundle cytokines are subdivided into short-chain and
long-chain cytokines, as their helices comprise approximately 15 or 25
residues, respectively (52, 53, 54). Crystal structures have been
determined for the long-chain four--helix bundle cytokines LIF (57),
IL-6 (61), CNTF (62), GH (63), granulocyte-colony stimulating factor
(G-CSF) (64), and leptin (65). Although exhibiting only a low degree of
homology in their primary structures, they show a high homology in
their tertiary structures and in their functional receptor epitopes
(66). The tertiary structure of LIF, from the N to the C terminus,
consists of helices A, B, C, and D, linked by two long loops AB and CD,
as well as the short loop BC (5, 55, 56, 57, 58, 59, 60). Three functional binding
sites, interacting with the LIFR and gp130 receptor subunit,
respectively, have been characterized (67, 68, 69) (Fig. 1A
).
|
|
III. LIF Receptor—Gene Structure and Regulation |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
-chain (IL-2, IL-4, IL-7, IL-9, IL-13, IL-15), or gp130
receptor subunit (LIF, OSM, IL-6, IL-11, CNTF, CT-1), respectively
(71). The LIFR—also referred to as low-affinity LIF receptor, LIFR,
LIFRß, or gp190—and the common gp130 receptor subunit both belong to
the class I cytokine receptor superfamily (72, 73, 74, 75).
B. LIFR gene and structure
The human (GenBank Accession X61615) (72), murine (GenBank
Accession D26177, S73496, S73495, S81861, X99778, X99779) (72, 76, 77, 78)
and rat (GenBank Accession D86345) (79) gene for LIFR have been
cloned. The LIFR gene is located on human chromosome 5p12–13 and
murine chromosome 15 within a cluster of cytokine receptor genes,
including IL-7, PRL, and GH receptor (80), suggesting ancestral
emergence from multiple gene duplications. The human LIFR gene spans
more than 70 kb and contains 20 exons (81). Alternative promoter usage
of the human LIFR gene yields a placental tissue-specific promoter
(GenBank Accession U78104) with a novel placenta specific enhancer
element, as well as an alternative promoter active in nonplacental
tissues (GenBank Accession AF018079) (82, 83).
C. Membrane-bound LIFR
Human LIFR (GenBank Accession X61615), is an
approximately 110-kDa protein that is glycosylated to about 190 kDa at
multiple potential N-linked glycosylation sites (72). Northern probe
analysis using a probe specific for human LIFR exhibits placental
mRNA transcripts of approximately 6.0 kb and 4.5 kb, as well as a minor
transcript of about 5.0 kb (72). Human LIFR preprotein is 1097 aa,
encompassing a signal sequence of 44 aa, an extracellular domain of 789
aa, a transmembrane domain of 26 aa, and a cytoplasmic domain of 238
aa. Human and murine LIFR share 76% amino acid sequence homology in
their extracellular domain (72, 79). The extracellular region of the
LIFR consists of two CBDs separated by an Ig-like domain, and three
membrane-proximal FBT III modules (69, 72) (Fig. 1B
).
D. Soluble LIFR
Murine LIFR exists in both a membrane-bound and a soluble form,
the latter lacking the transmembrane and cytoplasmic domains. In
Northern blot analysis for soluble or membrane-bound murine LIFR,
transcripts yield different sizes of approximately 3 kb or about 5 kb
and 10 kb, respectively (76, 77, 78). Both murine receptor forms are
derived from a single gene locus by alternative splicing. The cDNA of
membrane-bound murine LIFR (Gen Bank Accession D26177 and S81861) is
derived by alternative splicing, skipping an exon that is specific for
the soluble LIFR form, and contains a translation termination codon
(77). A B2 repetitive sequence, contained within the 3'-untranslated
region of soluble LIFR cDNA (GenBank Accession X99778), may cause
polyadenylation and regulate expression of soluble LIFR (77, 78).
Murine serum levels of soluble LIF-R are highest during pregnancy (84),
while a profound increase of soluble LIFR mRNA has been demonstrated in
liver during gestation (days 8–19), peaking at day 12 with an
approximately 20-fold increase (77). Alternative promoter usage for
transmembrane and soluble LIFR has been suggested, as a 5'-untranslated
exon 1 is expressed in most tissues, while an alternative exon 1a is
restricted to liver and, during gestation, is profoundly increased in
the liver and uterus (85). Murine soluble LIFR inhibits LIF action
in vitro and in vivo (86, 87), thus acting as an
important antiinflammatory modulator of LIF action. Recently, a human
soluble LIFR has also been described (81, 88) and shown to act as an
antagonist (88).
Soluble receptors, lacking their respective transmembrane and
cytoplasmic domains by alternative splicing events, have also been
identified for several other IL-6 family cytokines, including sIL-6R
(89, 90, 91), sIL-11R (92, 93, 94), and sCNTFR (95, 96). These soluble
receptor isoforms possess agonistic activity with their respective
ligand in cell lines expressing gp130, but lacking the membrane-bound
specific R subunit (89, 93, 94, 95, 96). On the other hand, antagonistic
effects have also been observed in other models (91, 94),
indicating a complex modulation of cytokine bioactivity by soluble
cytokine receptors.
E. gp130 Gene and structure
Human (GenBank Accession M57230) and murine (Genbank Accession
M83336, X62646) gp130 cDNAs have been cloned (73, 74). Human gp130 is
an approximately 100-kDa protein in its deglycosylated state, but
exhibits 14 potential N-linked glycosylation sites (73). Northern probe
analysis with a specific probe for the entire coding region of human
gp130 reveals ubiquitous gp130 mRNA expression with a single transcript
of approximately 7.0 kb (73). The human gp130 gene is located on
chromosome 5q11 (97, 98). Human gp130 preprotein is 918 aa, comprising
a 22-aa signal sequence, a 597-aa extracellular domain, a 22-aa
transmembrane domain, and a 277-aa cytoplasmic domain (73). The
extracellular gp130 domain consists of a N-terminal Ig-like domain, a
CBD, and three membrane-proximal FNT-III modules (73), thus exhibiting
structural similarity with the LIFR (Fig. 1B). The
cytoplasmic domain of LIFR and gp130 both contain three
homologous and functionally important motifs, termed box1, box2, and
box3 (3, 99). The crystal structure of the CBD of human gp130 has
recently been resolved (75).
Soluble forms of gp130 with a molecular mass of 90 to 110 kDa exist in human serum (100) and have been suggested to arise by proteolytic cleavage, rather than alternative splicing (101). Soluble gp130 has also been demonstrated to act as an antagonist of IL-6 (91, 100, 102) and LIF (101, 102) signaling, respectively.
F. IL-6 cytokine family and the gp130 receptor subunit
The IL-6 cytokine family is characterized by their receptors
sharing the common gp130 receptor subunit (2, 3, 4) and consists of LIF,
IL-6, IL-11, OSM, CNTF, and CT-1. Ligand binding of LIF, OSM, CNTF, or
CT-1 causes heterodimerization of gp130 with LIFR, and a third
cytokine-specific receptor subunit in the case of CNTF and CT-1 (2, 3, 4).
In contrast, ligand binding of IL-6 or IL-11 to their specific receptor
subunits does not involve LIFR and has been suggested to involve gp130
homodimerization (2, 3, 4), although other models will be discussed below.
Due to the shared receptor subunit and signaling cascade of all IL-6
cytokine family members, several of these cytokines show partially
overlapping or redundant hematological effects (2, 3, 4) (Fig. 2 and Table 1). Recently, two new members
of the IL-6 cytokine family, named cardiotrophin-like cytokine (103)
and neurotrophin-1/B-cell-stimulating factor-3 (104), have been
reported. Initial studies demonstrated cardiotrophin-like cytokine
(103) to involve tyrosine phosphorylation of gp130 and STAT1, and
neurotrophin-1/B-cell-stimulating factor-3 (104) to involve tyrosine
phosphorylation of gp130, LIFR, and STAT3 in their respective signaling
cascades.
|
|
1 x 10-9 M] to its specific
LIFR, subsequent association with gp130 forms a high affinity
(Kd 0.1 x 10-10
M) complex (72, 105).
LIF possesses three binding regions on distinct epitopes, similar to
CNTF, IL-6, and IL-11 (66, 67, 68, 69, 109). Mutagenesis analysis of each
cytokine revealed site I to bind with the specific cytokine receptor,
while site II binds to gp130 (66, 67, 68, 69, 109). Binding site III has
various functions, as it binds to the LIFR in the case of LIF (67, 68, 69),
but allows contact with a second gp130 molecule in the case of IL-6 and
IL-11 (67, 68, 109, 110, 111, 112, 113). In an electrostatic analysis model, derived
from the crystal structure (57, 58) and mutagenesis studies of LIF (67, 68, 114), LIF binds to the membrane-proximal CBD of LIFR (site I) and
to the CBD of gp130 (site II) (69) (Fig. 1B). While some data suggest
that LIF binding site III binds to the membrane-distal CBD of the LIFR
(69), others have found the Ig-like domain of the murine LIFR to be
essential for high-affinity LIF binding (115, 116). Based on LIFR
mutagenesis studies, two distinct LIF binding sites in the
membrane-distal CBD and Ig-like domain have also been proposed, while
the interacting membrane-proximal CBD was suggested to be important for
protein conformation (116). Phe156 and Lys159, located in site III at
the N-terminal end of the D helix, are important residues for binding
to the LIFR and are conserved in LIF, OSM, CNTF, and CT-1 (68), all of
which bind to the LIFR.
H. The LIFR-gp130 complex signals OSM, CNTF, and CT-1
In addition to LIF signaling, the LIFR and gp130 heterodimer is
also required for signal transduction of OSM, CNTF, and CT-1 (2, 3, 4).
Signaling of OSM is achieved by either a heterodimer of the common LIFR
and gp130 (OSM receptor type I) or the specific OSMR and gp130 (OSM
receptor type II), respectively (117, 118, 119, 120, 121, 122). While human OSM activates
OSM type I receptors to a similar extent as does LIF (120), murine
(m)OSM exhibits a 30- to 100-fold lower binding and no activation of
the OSM receptor type I (120, 121, 122). In contrast, mOSM specifically
activates only the OSM receptor type II (117, 120, 121, 122). CNTF signaling
is mediated by a tripartite complex of CNTFR, LIFR, and gp130
(123, 124, 125, 126). CT-1 also requires the LIFR and gp130 for signaling
(127, 128, 129) and has been suggested to form a tripartite receptor complex
similar to CNTF, including LIFR, gp130, and a glycosylated 80-kDa
protein (128).
I. IL-6:IL-6R and IL-11:IL-11R complex
In contrast to LIFR-mediated signaling of LIF, OSM, CNTF, and
CT-1, the LIFR is not involved in signaling of IL-6 and IL-11. Both
IL-6-IL-6R (130, 131, 132, 133, 134, 135, 136) and IL-11-IL-11R (137, 138, 139) require gp130 for
complex formation, recognize two distinct binding motifs on gp130, and
compete for binding to gp130 (111, 112). In different models (2, 3, 4, 61, 111, 112, 130, 131, 132, 133, 134, 135, 136), IL-6 binds to its specific IL-6R subunit, causing
either a hexameric complex consisting of IL-6:IL-6R:gp130 in 2:2:2
formation, or homodimerization of gp130 in a tetrameric complex. A
similar model of gp130 homodimerization has been proposed for IL-11
(2, 3, 4, 109, 111, 112); however, monomeric gp130 further enables a
pentameric complex that consists of two of each IL-11 and IL-11Ra
(139).
J. LIF binding and LIFR expression
Low- and high-affinity binding sites for LIF have been described
in several cell types (72, 140, 141). A low number of approximately
150–400 high-affinity binding sites with a Kd of
10 - 200 x 10-12 M is found on
most cells responsive to LIF. Furthermore, approximately 1,000–6,000
low-affinity binding sites with a Kd of 1–4
x 10-9 M are present on many cell
types. While LIFRa constitutes the low-affinity binding site,
association of the LIF-LIFR complex with gp130 results in its
conversion to a high-affinity binding site (72, 105).
A recent study demonstrated the mannose-6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) to be a nanomolar affinity receptor for glycosylated, but not for deglycosylated, human LIF (142, 143). Several human cell lines exhibiting no detectable binding of nonglycosylated human LIF, revealed 3,000 to 40,000 binding sites for glycosylated human LIF, due to the Man-6-P/IGFII-R (142). Therefore, low-affinity binding of glycosylated human LIF seems to be not only mediated by the low-affinity receptor LIFR, but also to a large extent by the Man-6-P/IGFII-R. Binding of LIF to the Man-6-P/IGFII-R caused no downstream functional effects, but mediated a rapid internalization and degradation of LIF (143). Therefore, the Man-6-P/IGFII-R might regulate LIF bioavailability (143).
|
IV. LIF Signaling |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
B. Jaks
Jak 1 and Jak 2 associate with the cytoplasmic receptor
subunits gp130 and LIFRß in the absence of ligand, but are
autophosphorylated and activated only after ligand binding and
heterodimerization of the LIFR-gp130 complex (149) (Fig. 1B).
Heterodimerization of the LIFR-gp130 complex by LIF activates Jak1
(150, 151, 152, 153), Jak2 (151, 152, 153), and Tyk2 (151) kinase activity, followed
by phosphorylation of gp130 and the LIFR (150, 151, 154, 155). Targeted
disruption of the Jak1 gene abrogates gp130-mediated signaling (156),
while targeted disruption of the Jak2 gene does not abolish LIF or IL-6
responsiveness (157, 158). Similarly, in vitro transient
overexpression of a dominant negative Jak1 mutant almost completely
abrogated LIF responsiveness, while a dominant negative Jak2 mutant
attenuated LIF signaling only by approximately 30% (159). All these
data suggest an essential role of Jak1 for LIF signaling.
C. STATs
Phosphorylated tyrosine residues on LIFR and gp130 provide
specific docking sites for the SH2-domains of STAT proteins (149, 160, 161, 162), causing receptor association and subsequent phosphorylation
of STAT1 (128, 150, 159, 160, 161, 162, 163, 164) STAT3 (128, 150, 151, 153, 154, 155, 163, 164, 165, 166, 167, 168, 169), or STAT5a (170, 171), respectively. The pattern of Jak/STAT
protein activation by LIF is cell type specific (150). STAT1(-/-)
embryonic stem cells derived from mice with targeted disruption of the
STAT1 gene are no longer responsive to interferons (IFNs), but still
respond to LIF (172). However, overexpression of STAT3 dominant
negative mutants (173, 174) or lowering of activated STAT3 levels (175)
inhibits LIF-induced maintenance of pluripotent embryonic stem cells.
Similarly, overexpression of STAT3 dominant negative mutants also
inhibits LIF-induced differentiation of leukemic M1 cells (176), POMC,
and SOCS-3 expression of corticotroph AtT-20 cells (177, 178), as well
as c-fos and atrial natriuretic factor expression in
cardiocytes (150). These results demonstrate a compelling role of
STAT-3 for LIF-signaling in several cell types (Fig. 1B).
D. Cytoplasmic receptor domains
Using chimeric receptor models, homodimers of gp130 as well as
LIFRß were shown to be sufficient for STAT3 tyrosine phosphorylation
(179). Carboxy-terminal truncation of the cytoplasmic gp130 or LIFRß
domain, respectively, revealed that the membrane-proximal box 1 and box
2 regions are not sufficient for STAT3 phosphorylation (161, 152, 179).
In contrast, the 74 membrane-proximal aa of the LIFRß are sufficient
for binding of Jak1 and Jak2 (149). Further mutagenesis analysis
demonstrated a consensus sequence YXXQ, located on several
membrane-distal locations in the cytoplasmic domains of gp130 and
LIFRß, respectively, which is required for STAT3 association with the
receptor and subsequent phosphorylation (Fig. 1B). Thus, tyrosine
phosphorylation of the YXXQ motif provides a binding motif for the
highly specific SH2-domain of STAT3 (160, 162, 179). Binding of STATs
to the cytoplasmic receptor subunit causes a closer steric association
with Jak kinases, which may result in STAT tyrosine phosphorylation.
Phosphorylation of C-terminal tyrosine sites in STAT3 (Tyr 705) and in
STAT 1 (Tyr 701) causes the SH2 domains to enable homo- or
heterodimerization of STAT-3-STAT3, STAT1-STAT3, or STAT-1-STAT1,
respectively (146, 147). Crystal structure analysis of STAT3 and STAT1
homodimers demonstrates that the SH2 domain of a STAT monomer binds to
the C-terminal phosphotyrosine of the other, thus enabling
homodimerization (180, 181). The dimerized STAT complexes are
translocated to the nucleus, and their DNA-binding domain (aa 400–500)
binds to specific DNA STAT-binding elements (SBE), causing
transcriptional activation (146, 147, 182) (Fig. 1B). In addition to
primary tyrosine phosphorylation, IL-6 cytokine family members also
cause secondary serine phosphorylation of STAT3 and STAT1 (147, 169, 183, 184, 185, 186, 187). Secondary serine phosphorylation of STATs has been
controversially shown to either enhance DNA binding of STAT3-STAT3
complexes (183) or to have no effect (147, 169, 186, 187). However,
despite not directly effecting DNA binding of STAT3 complexes, serine
phosphorylation of STAT3 seems to be required for full transactivation
of STAT-responsive genes (147, 169, 186, 187).
E. Negative feedback regulators of Jak-STAT signaling
Negative feedback regulators of gp-130-mediated activation of
Jak-STAT signaling include the tyrosine phosphatase SHP-2, as
well as members of the newly described SOCS and PIAS protein families.
Those negative feedback regulators negatively interfere with the
LIF-induced Jak-STAT signaling cascade at different levels (Fig. 1B).
F. SHP-2
The SH2-containing protein tyrosine phosphatase-2 (SHP-2) is a
cytosolic protein involved in regulation of tyrosine kinase-mediated
signaling pathways (for review see Refs. 188, 189, 190). LIF stimulates
tyrosine phosphorylation of SHP-2 (191) by a Jak-1-dependent pathway
(192). After LIF stimulation, SHP-2 associates with the cytoplasmic
gp130 receptor subunit (191). A membrane-proximal tyrosine
phosphorylation site in the cytoplasmic domain of the gp130 receptor
(Y118) is essential for tyrosine phosphorylation of SHP-2 (179, 193, 194, 195). Overexpression of dominant negative SHP-2 variants (194, 196, 197) or a mutated gp130 subunit lacking the cytoplasmic binding
site for SHP-2 (194) significantly enhanced CNTF- or LIF-induced
effects in different cell models. Therefore, a negative feedback
regulation of SHP-2 on gp130-mediated STAT activation has been
suggested.
G. SOCS proteins
SOCS proteins are a new family of proteins termed suppressors of
cytokine signaling (SOCS), STAT-induced STAT inhibitors (SSI),
cytokine-inducible SH2 containing protein (CIS), and Jak-binding
protein (JAB). Several current reviews have summarized the fast growing
knowledge on this protein family (198, 199, 200, 201). SOCS-1 and/or SOCS-3 can
inhibit the signaling cascade of several Jak-STAT-dependent cytokines,
including the gp130 sharing cytokines LIF (202, 203, 204, 205, 206, 207), IL-6 (202, 208, 209, 210), OSM (202), and CNTF (211), as well as GH (212), PRL (213),
leptin (214, 215), IL-4 (216, 217), and IFNs (218, 219). Overexpression
of SOCS-3 inhibits LIF-induced phosphorylation of gp130 and STAT3, as
well as STAT3-mediated downstream events (206). Recent studies revealed
SOCS-1 to inhibit Jak2 activity by binding to the catalytic JH1 domain
of Jak2 (204, 207, 208, 209, 210). A similar mechanism of Jak-STAT inhibition has
also been suggested for SOCS-3 (209, 215, 220), while others suggested
a slightly different mechanism with no direct inhibition of Jak kinase
activity (221). SOCS protein expression is stimulated by multiple
cytokines in a tissue- and cell type-specific manner (198, 199, 200, 201, 202). As
both SOCS-1 (203) and SOCS-3 (178) gene expression have been
demonstrated to be STAT-3 dependent, there exists a negative
autoregulatory feedback of SOCS-1 and SOCS-3 on their own gene
expression. In addition, a recent study also found STAT-independent
induction of SOCS-3 gene expression by IL-10 (222), while we observed
induction of SOCS-3 by IL-1ß, which was not mediated by the -72 to
-64 STAT-RE in the SOCS-3 promoter (our unpublished results).
H. PIAS
Another family of negative regulators of STAT signaling, termed
PIAS, has recently been described (223, 224). In contrast to SOCS
proteins inhibiting Jak activity and subsequent STAT phosphorylation
and activation, PIAS1 and PIAS3 interact directly with both activated
STAT-1 and STAT3, respectively, and inhibit their binding to specific
DNA sequences.
I. Mitogen-activated protein kinase (MAPK)
In addition to activating the Jak-STAT cascade, several IL-6
cytokine family members also stimulate the Ras-MAPK pathway (2, 3, 4, 5, 144, 145) (Fig. 1B). In the Shc/Grb2/SOS/Ras/Raf/Mek/Erk signaling cascade,
serine/threonine kinases Erk1/2 themselves activate numerous nuclear
transcription factors, as well as cytosolic and cytoskeletal targets
(for review see Refs. 144, 145, 225).
LIF has been demonstrated to stimulate Shc (152), Ras (152, 226), Raf-1
(227), MAPKK (228), as well as Erk1 and Erk2 activity (141, 150, 152, 165, 168, 173, 227, 228, 229). The ability of LIF to induce MAPK tyrosine
phosphorylation and activity is cell-type specific (141, 152, 168, 177, 227, 228, 229) and is probably essentially required for distinct LIF
activities, while it is not required for others (152, 161, 177). SHP-2
has been shown to be essential for LIFR/gp130-mediated activation of
MAPK (161, 192, 229, 230), despite its inhibitory function on
gp130-mediated STAT activation (194, 196, 197). As discussed above,
LIF-induced SHP2 activation requires specific cytoplasmic tyrosine
residues on gp130 (Y118) and LIFR (Y115) (161, 179, 193, 194, 195). Deletion
of these essential tyrosine residues or coexpression of a
dominant-negative SHP2 mutant blocks subsequent MAPK activity (161, 229). These data suggest LIFR/gp130-stimulated MAPK activity to be
mediated through activation of SHP-2 (Fig. 1B). Phosphatidylinositol
(PI) 3-kinase also seems to be an essential mediator of LIF- and
IL-6-induced MAPK activation (230, 231), as the PI-3-kinase inhibitor
wortmannin inhibits LIF- and IL-6-induced activation of MAPK activity,
while STAT3 phosphorylation was mostly unaffected.
Bidirectional interactions of the Jak-STAT and the Ras-MAPK pathway are suggested by several lines of evidence. The LIFR itself is a target of LIF-induced MAPK activity and is phosphorylated on Ser-1044 in its cytoplasmic domain (229). Whether secondary serine phosphorylation of STAT3 (183, 184) depends on MAPK activity is controversial (167, 169, 185, 232). The biological significance of secondary serine phosphorylation of STATs is also still controversial and might differ among cell types (169, 183, 184, 185, 233). Recently, activation of Erk1/2 has been demonstrated to inhibit Jak1 and Jak2 kinase activity, while serine phosphorylation of STAT3 did not play an essential role (233). Based on these data, a close interaction of the Jak-STAT and Ras-MAPK pathways is now apparent. Further studies are needed to understand these interactions.
J. Others
1. Insulin receptor substrate (IRS). IRS proteins are adaptor
proteins with multiple tyrosine phosphorylation sites, serving as
docking sites for SH2-domains of various proteins. IRS proteins are
involved in signaling of insulin and various cytokines (for review see
Refs. 145, 234). In 3T3-F442A fibroblasts, LIF stimulates tyrosyl
phosphorylation of IRS-1 (235) and IRS-2 (236), respectively.
Phosphorylated IRS-1 or IRS-2 associates with various proteins,
including p-85 regulatory subunit of phosphatidylinositol 3'-kinase,
Grb-2, or protein tyrosine phosphatase SHP-2, respectively (145, 234).
As these molecules have been ascribed to involvement in LIF signaling,
IRS proteins likely play a modulatory function in the LIF signaling
cascade and therefore merit further investigation.
2. Tyrosine kinases. In addition to the Jak-STAT signaling cascade, several other cytoplasmic tyrosine kinases are activated by IL-6, including Btk (237), Tec (237), Fes (238), p59Fyn (239), p56/59Hck (239), and p56Lyn (239). Because of largely overlapping actions in the IL-6 cytokine family, there might also be a potential role for some of these kinases in the LIF signaling pathway. So far, to the best of our knowledge, only LIF-induced activation of Hck has been reported (152, 240). Therefore, further investigation should elucidate the potential involvement of these cytoplasmic kinases in LIF signaling.
|
V. LIF—Hematopoietic and Neuropoietic Cytokine |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
LIF was first cloned in 1987 and characterized by its ability to stimulate differentiation of the murine myeloblastic leukemia cell line M1 (7). Thereafter, numerous studies used M1 cells as an in vitro model for studying LIF binding on LIFR/gp130 complex, intracellular mechanisms of LIF action (171, 176), and regulation of the Jak STAT-induced signaling cascade by SOCS proteins. Studies overexpressing SCL (242, 243), flt3 ligand (244), or Wilm’s tumor suppressor gene (245) in M1 cells have partially enlightened the downstream events of LIF-induced M1 cell differentiation.
Due to the close similarities of the gp130-related IL-6 cytokine
family, several of these cytokines show partially overlapping or
redundant hematological effects (2, 3, 4) (Table 1). Animal models have
confirmed important involvement of gp130-related cytokines on the
hematopoietic system. Targeted disruption of gp130 causes death of
homozygous murine embryos (gp130 -/-) between 12.5 days postcoitum
(pc) and term, because of cardiac hypoplasia and hematological
disorders with greatly reduced fetal liver pluripotential and committed
hematopoietic progenitor cells (246). Postnatally induced inactivation
of gp130 resulted in a less pronounced decrease in hematopoietic
progenitor cells, while in the peripheral blood, reduced platelet
counts was the most striking finding (247). Homozygous LIF knockout
mice (LIF -/-) have reduced numbers of pluripotent hematopoietic stem
cells in spleen and bone marrow and impaired thymic maturation (248),
indicating an important role of LIF in hematopoietic stem cell
survival/proliferation. Strikingly, homozygous LIFR knockouts (LIFR
-/-) did not reveal major hematological abnormalities and showed
normal colony formation of pluripotent hematopoietic progenitor cells
(249). Taken together, these animal models demonstrate that
gp130-related cytokines, including LIF, are essential for
hematopoiesis.
Although devoid of intrinsic proliferative action, LIF acts as a hematopoietic growth factor that synergistically costimulates hematopoietic progenitor cell proliferation (241, 250, 251). Another indirect mechanism of LIF action on hematopoietic stem cells is mediated by LIF-induced upregulation of stromal bone marrow-derived cytokines (252). LIF stimulates megakaryocyte proliferation and platelet production (241, 253, 254, 255, 256, 257) and specifically induces proliferation of IL-3-stimulated murine and human megakaryocytes in vitro (241, 253). In vivo experiments in mice (241, 254, 255) and primates (256, 257) demonstrate that daily LIF administration for 1–2 weeks causes an approximately 2-fold increase in circulating platelet levels. Recent studies also suggest costimulatory effects of LIF on murine and human erythroid (258, 259) and macrophage (260, 261) progenitor cells.
In addition to IL-6, other members of the IL-6 cytokine family, including LIF, also play an important role in stimulating survival and proliferation of multiple myeloma cells. This topic has been recently reviewed (262, 263). LIF stimulates myeloma cell growth, probably acting as a paracrine growth factor (107, 264).
B. Nervous system
LIF has been termed a cytokine at the interface between
neurobiology and immunology (265); in addition to its effects on the
hematopoietic system, various neuropoietic effects (265, 266),
e.g., switching of sympathetic neuronal phenotype and rescue
and differentiation of sensory and motor neurons as well as glia cells,
have been demonstrated. This manuscript will only briefly discuss
recent knockout animal studies, elucidating the physiological roles of
LIF, LIFR, and gp130 in the nervous system. For extensive information
on LIF- and LIFR-mediated effects in the nervous system, we recommend
the comprehensive review provided by Murphy et al. (266).
LIF stimulates cholinergic differentiation of sympathetic neurons in vitro (267) and in vivo (268). However, studies on mice deficient in LIF or CNTF demonstrated that neither LIF nor CNTF is essential for cholinergic differentiation of sweat gland sympathetic neurons (269). On the other hand, in vitro blockade of LIFR in neuron/gland cocultures inhibited cholinergic differentiation activity (270). These data demonstrate that cholinergic differentiation of sympathetic neurons requires LIFR activation, while LIF and CNTF can act as stimuli. However, as LIF- and/or CNTF-knockout mice show intact cholinergic differentiation of sympathetic neurons, other cytokines acting through the LIFR can probably compensate for their deficiency.
Gene knockout studies have demonstrated differentiation of astrocytes and expression of the astrocyte marker glial fibrillary acidic protein (GFAP) to be mediated by LIFR (249, 271, 272) and gp130 (273, 274). Furthermore, stimulation of astrocyte differentiation is dependent on the Jak-STAT pathway (271, 275), as shown by mutation analysis of chimeric cytoplasmic gp130 or LIFR components and overexpression of STAT3 dominant negative mutants (271). In LIF knockout animals, decreased numbers of GFAP-positive cells are found in the hippocampus (273, 276). These data indicate LIF to be essential for differentiation of astrocytes in certain brain areas. However, other cytokines acting through the LIFR-gp130 receptor complex might compensate for most neurotrophic LIF actions.
|
VI. LIF and Endocrine Systems |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
1. Knockout models and implantation. Striking evidence for the essential role of LIF in embryonic implantation was provided by Stewart et al. (283), who showed that female LIF knockout LIF-/LIF- mice are infertile, due to a defect in endometrial decidualization and embryonic implantation. Blastocyst transfer from female LIF-/LIF- mice to pseudopregnant wild-type (wt) LIF+/LIF+ mice resulted in implantation and successful pregnancies. Treatment of LIF-/LIF-mice with recombinant LIF also enabled successful implantation. Thus, in the mouse, LIF plays an obligatory role in embryonic implantation. The requirement of LIF for successful murine implantation seems to be similar for other mammals, as passive immunization of ewes and cows against LIF results in a reduced pregnancy rate (284). Due to multiple systemic abnormalities, both the LIFR-/LIFR- (249) and gp130-/gp130- /(246) knockout models are not viable beyond term, and therefore implantation in female knockout animals cannot be studied in these models.
Recently, the essential role of IL-11, another member of the IL-6 cytokine family, has been demonstrated for successful decidualization. Robb et al. (285) demonstrated female IL-11Ra-/IL-11Ra- knockout mice to be infertile due to defective decidualization. This lesion appeared isolated, as the female IL-11Ra-/IL-11Ra- mice exhibited otherwise normal estrous cycle, oozyte fertilization, and blastocyst development. Male IL-11Ra-/IL-11Ra- mice are normally fertile. As members of the IL-6 cytokine family exhibit partially overlapping and redundant functions, these findings resemble the results in LIF-/LIF- knockout mice (283). However, RNAse protection analysis showed a distinct temporal pattern of decidual cytokine expression, indicating a cascade of different events. While decidual LIF expression peaked on days 2.5 to 3.5 pc and rapidly declined thereafter, IL-11 expression peaked at days 5.5 to 7.5 pc (285). Thus, despite partially overlapping functions in promoting uterine decidualization and blastocyst implantation, LIF and IL-11 appear to act in a specific temporal cascade in the uterus.
2. Uterine LIF expression. Uterine LIF expression in adult virgin mice is barely detectable during diestrous, proestrous, and metestrous II (286), but peaks during estrous and metestrous I, corresponding to late endometrial proliferative and early secretory phases, including ovulation (286). During early pregnancy, uterine LIF expression at day 0.5 pc is similar to that found during estrous and metestrous I. At day 1.5 and 2.5 pc, uterine LIF expression declines, but peaks at day 3.5 pc shortly before implantation of the blastocyst. After implantation at day 4.5 pc, uterine LIF expression then declines again, becoming nearly undetectable (286). Thus, peak endometrial LIF expression occurs early, preceding or coinciding with the time of blastocyst implantation, in the mouse (286, 287, 288), rat (289), rabbit (290), pig (291), mink (23), and western spotted skunk (292). In contrast, in sheep endometrial LIF expression is relatively constant throughout the estrous cycle and early pregnancy (293), and in pseudopregnant mice (287) and rabbits (290) the pattern of uterine LIF expression is similar to that observed in pregnant animals. These data indicate endometrial LIF expression to be under maternal control, independent of stimuli from the conceptus.
In humans, LIF mRNA and protein are maximally expressed in endometrial samples derived from normal cycling women in the mid and late secretory phase (45, 294, 295, 296, 297, 298). Glandular and luminal epithelial cells account for the majority of endometrial LIF mRNA and protein (45, 294, 297, 298, 299). Immunohistochemical studies show luminal and glandular epithelial LIF staining to be cycle dependent, peaking during the mid and late secretory phase (294, 297, 298, 299). In contrast, during the proliferative phase, luminal and glandular epithelial LIF mRNA and protein were not at all (294, 298) or only faintly (297, 299) expressed. Results of immunohistochemical studies of LIF expression in stromal cells are controversial. Stromal cells consist mostly of fibroblasts and leukocytes (300). While some studies report modest LIF expression in endometrial stromal cells only during the proliferative (299) or secretory phase (294), respectively, others found stromal LIF constantly expressed throughout the menstrual cycle (292). Thus, human endometrial luminal and glandular cells are the major contributor of endometrial LIF mRNA and protein expression and show a cycle-dependent peak of LIF expression in the mid and late secretory phase. This is also the timepoint when blastocyst implantation occurs and suggests an important role for LIF in human implantation, as has been demonstrated in the LIF knockout mouse (283).
In vitro, a permanent human epithelial endometrial cell line
has been shown to produce small amounts of LIF (291). Explant cultures
from human endometrial glandular epithelial cells exhibit significantly
higher LIF mRNA expression and secretion than do stromal cells (45, 296, 301, 302). In vitro LIF expression by stromal cells was
also induced by IL-1, TNF, platelet-derived growth factor (PDGF),
epidermal growth factor (EGF), and transforming growth factor-ß
(TGFß), while interferon- inhibits LIF expression in these cells
(45). Similarly, LIF secretion from first-term decidual cells is
stimulated by IL-1, TNF, and TGFß (303).
3. LIF effects on decidual cell cultures. During pregnancy, decidual cells as well as cytotrophoblasts express LIF mRNA and protein. Decidual culture explants from pregnant women show significant LIF production and secretion (304, 305), which correlates with the pregnancy duration (304). High levels of LIF are encountered during the first trimester and at term, but lower LIF secretion occurs during the second trimester (304). LIF secretion by decidual explants derived from women with early ectopic pregnancy between days 35 to 76, all showed high levels of LIF secretion, irrespective of the pregnancy term (305). A stimulatory effect of estradiol on LIF secretion was observed in this primary culture model (305).
Serum levels of LIF are lower in pregnant women in comparison to nonpregnant women (306), while serum levels of soluble LIF-R increase severalfold during pregnancy in mice (84) and humans (306). Although the significance of these findings is not yet understood, locally produced uterine LIF seems to act in an autocrine/paracrine fashion rather then systemically. The soluble LIF-R, however, might act as a negative regulator (84, 306), modulating local as well as systemic LIF actions.
4. Hormonal regulation of uterine LIF expression. Hormonal regulation of endometrial LIF expression is not fully understood. Data on possible effects of estrogen or progesterone on endometrial LIF expression are contradictory. Comparison of in vivo studies in different species is complicated by variation of implantation type, modus, and hormonal regulation (281, 307). In vitro, primary endometrial cell cultures are derived from different sources, including different cell subtype enrichments, primary cultures started at different stages of the ovulatory cycle, or early pregnancy, respectively.
Implantation in mice is dependent on estrogens, and endometrial LIF protein expression in ovariectomized mice is up-regulated by estrogen, while progesterone has no stimulatory effect (307). Mixed monolayers derived from whole murine uteri at day 3 pc exhibited expression of diffusible LIF in an RNAse protection assay (308). However, expression of diffusible LIF was not altered by estrogen, progesterone, or a combination of estrogen plus progesterone treatment (308). In contrast to mice, rabbits are not dependent on maternal estrogens for implantation, and endometrial LIF protein expression is up-regulated by progesterone, while estrogen has no effect (307). In ovariectomized ewes, both estrogen and progesterone had an inhibitory effect on endometrial LIF expression (293).
Although LIF expression in human glandular endometrial epithelial cells is highest during the progesterone-driven secretory phase (45, 294, 295, 296, 297, 298), data on the effects of estradiol and progesterone on LIF expression are incongruent, which in part might be explained by tissue- and phase-specific effects. Reporter gene activity of a human LIF promoter luciferase construct was stimulated 3.5- to 7-fold by medroxyprogesterone acetate in uterine tumor SKUT-1B cells, cotransfected with progesterone receptor A or B, respectively (309). Treatment of fertile women with 200 mg of the progesterone antagonist, mifepristone, on day LH+2 resulted in a decreased LIF expression in glandular endometrial epithelial cells on day LH+6, while the steroid had no effect on luminal epithelial or stromal cells (310). In contrast, in a primate model, treatment of rhesus monkeys with 2 mg/kg mifepristone on day LH+2 had no effect on endometrial LIF expression on day LH+6 (311). In vitro, estradiol, progesterone, and medroxyprogesterone acetate have been reported to lack an effect on endometrial stromal cells (45). These data would explain why stromal LIF expression is not cycle dependent (294, 297, 299). LIF expression in epithelial endometrium explant cultures slightly decreases during incubation with estradiol and progesterone (302). ßhCG did not show an in vitro effect on LIF expression of mixed endometrial cells derived from women undergoing oocyte retrieval for in vitro fertilization (IVF) (312).
5. Uterine LIFR and gp130 expression. Similar to uterine LIF expression during early pregnancy, expression of LIFR and gp130 in the endometrium is up-regulated during early pregnancy (288, 313, 314). LIF binding and gp130 immunoreactivity peak on days 3 and 4 of mouse pregnancy (288) and days 5 and 6 of rabbit pregnancy (313), while blastocyst implantation takes place on day 3.5 pc in the mouse and day 7 pc in the rabbit, respectively. Using in situ hybridization, murine LIFR and gp130 mRNA expression were detected in decidual tissue, with highest expression evident in decidua directly surrounding the embryo (314). By day 8.5 pc, LIFR expression decreased and was only detectable in a small area near the placenta, while gp130 mRNA increased in the whole decidua beyond day 8.5 pc. Northern blot analysis revealed a 3.0- and 10.0-kb decidual LIFR transcript, compatible with the soluble and membrane-bound form of the murine LIFR (314). From preimplantation to day 8.5 pc, LIFR and gp130 mRNA was also expressed in uterine endometrial glands (314). Several possible functions of LIFR and gp130 in the murine decidua have been proposed (314), including the notion that LIF may act directly, regulating decidual growth and maturation.
Using Northern blot analysis of total RNA, human LIFR could not be detected in endometrial samples derived during the proliferative or secretory phase (298, 315), while gp130 was low but constitutively expressed, peaking during the secretory phase (315). Low LIFR mRNA expression was found in first trimester decidua, while chorionic villi of the first trimester exhibited high expression of LIFR mRNA (315). Using in situ hybridization, human LIFR was found to be expressed in the luminal epithelium of the endometrium, but not in glandular epithelium or stromal cells (298). gp130 Was detected in luminal as well as glandular epithelium (298).
6. LIF in follicular fluid. LIF is present in human follicular fluid from women undergoing IVF and embryo transfer (316, 317, 318). LIF levels in follicular fluid were significantly higher in preovulatory follicles derived from women after ßhCG treatment, as compared with immature follicles derived from women before ßhCG treatment (316, 317). Cultured ovarian stromal cells (316) as well as granulosa-lutein cells (316, 317) exhibit low constitutive LIF expression. Furthermore, cultured granulosa cells derived from mature follicles exhibit increased LIF production after ßhCG stimulation, while granulosa cells from immature follicles do not respond to ßhCG (317). Thus, LIF might be involved in ovulation and/or final oocyte development. However, no correlation between LIF levels in follicular fluid and IVF outcome could be established (318), and LIF-/LIF- mice exhibit normal blastocyst formation rates before implantation (283). Therefore, LIF does not appear to be essential in this process, or its loss might be compensated by other factors.
7. Embryonic LIF, LIFR, and gp130 expression. By RT-PCR, LIF mRNA is first detectable in the murine embryo at the morula stage, while LIFR and gp130 mRNA are first detectable at the blastocyst stage (314). In situ hybridization reveals a distinct localization of LIF expression in the trophoectoderm, but not in the inner cell mass, while LIFR and gp130 are primarily localized in the inner cell mass (314). This expression pattern is highly suggestive of a paracrine regulation of the inner cell mass by trophoectodermal-derived LIF (314).
In vitro, cultures of mouse embryos for 7 days in LIF-supplemented medium resulted in an approximately 40–50% increased inner cell mass and trophoblastic area, in comparison to embryos cultured in nonsupplemented medium (308). In vitro, supplementing culture media with LIF increases the percentage of murine eight-cell embryos to develop beyond the hatched blastocyst stage, to hatch or exhibit trophoblast outgrowth in vitro (308).
Human morula- and blastocyst-stage embryos also express gp130 mRNA and LIFR mRNA (295, 319, 320). Furthermore, gp130 (321) and LIFR (322) are expressed on cytotrophoblasts.
LIF had no effect on cell proliferation or expression of integrins
1, 5, or ß1 by cultured human trophoblast cells (322). However,
LIF affects cytotrophoblast ßhCG and fibronectin production in a
phase-specific manner, although the available data are discordant. LIF
increases ßhCG production of cytotrophoblasts derived from first
trimester placentas (304, 322), while it decreases ßhCG production of
cytotrophoblasts purified from placentas of term pregnancies (304, 323). In contrast, another study reported a decrease of ßhCG
production in cytotrophoblasts derived from first trimester (324). LIF
also inhibits forskolin- and cAMPinduced ßhCG production by the
human choriocarcinoma cell lines BeWo (313) and JEG-3 (304). LIF also
increases fibronectin expression by cytotrophoblasts purified from
placentas of term pregnancies (323), while no effect was observed in
cytotrophoblasts derived from first trimester placentas (324).
8. LIF and embryonic stem cells. LIF is essential for inhibition of pluripotent embryonic stem cell differentiation (152, 161, 173, 174, 175, 197, 308, 325, 326, 327, 328, 329, 330) and promotion of primordial germ cell growth by inhibiting apoptosis (331, 332, 333, 334, 335, 336). These features attribute LIF an essential factor for in vitro maintenance and growth of pluripotential ES cells and PG cells. For inhibition of pluripotent embryonic stem cell differentiation by LIF, STAT3 activation is essential, while SHP2 and MAP kinase activation are not required (330). Interestingly, recent data demonstrate that some pluripotent ES cells may be LIF independent (337, 338). This LIF-independent alternative pathway of pluripotent ES cell maintenance provides a possible explanation why gp130 (246) and LIFR (249) could be successfully targeted to generate knockout mice. Also, the primordial germ cell compartment was not affected in LIFR knockout mice (249), indicating the presence of an alternative developmental pathway. LIF action on ES and PG cells is important for in vitro research, as well as laboratories interested in IVF or gene targeting procedures.
9. Uterine LIF and immune cells. The action of LIF as an "interplayer" between the immune and endocrine systems has recently been reinforced by a study demonstrating LIF production by decidual T cell leukocytes (322, 339) producing also the TH2 cytokine subset (IL-4, IL-10) (339). Progesterone stimulates T cell LIF production via IL-4 (339). Production of LIF, IL-4, and IL-10 was significantly reduced in decidual CD4+ cell clones derived from women with unexplained recurrent abortions in comparison to control fertile women, while no differences were observed in the respective peripheral blood T cells of both groups (339). Similarly, another study reported that while decidual CD56+CD16- NK cells and CD3+ T cells expressed LIF, no LIF expression was evident in the same peripheral blood leukocyte subsets (340). These results indicate an important microenvironmental role of decidual CD4+ cells as mediators of paracrine LIF secretion. This hypothesis is also strengthened by a recent animal study (341) reporting that intravenous administration of CD4+ thymocytes in pseudopregnant mice increased uterine LIF mRNA expression and successful implantation rate after blastocyst transfer.
10. LIF and failure of implantation. Although the role of LIF in human embryonic implantation is not totally resolved, recent studies have linked some cases of unexplained infertility to an altered pattern of endometrial LIF expression (302, 339, 342, 343) or to LIF gene mutations (344), possibly causing decreased availability or biological activity of LIF in the uterus. In two studies, endometrial explant cultures derived from women with unexplained infertility and multiple implantation failures demonstrated reduced LIF expression in the secretory phase to approximately 30–40% of LIF secretion by control fertile women (342, 343). Another study examining LIF content in uterine flushings showed that on day LH+10, intrauterine LIF was lower in samples obtained from women with unexplained infertility than from control fertile women (302). As mentioned above, production of LIF, IL-4, and IL-10 was reduced in decidual CD4+ cell clones derived from women with unexplained recurrent abortions in comparison to control fertile women (339). In a study examining nulligravid infertile women (n = 74) and fertile controls (n = 75), heterozygous point mutations in the coding-region of the LIF gene were found in three infertile women, while only one point mutation/polymorphism in the non-coding region was observed in the fertile women (344). During the proliferative phase (cycle day 10), endometrial LIF concentrations correlate negatively with sonographic endometrial thickness (345), suggesting a role for LIF not only in the secretory phase, but also in the proliferative phase of the cycle. These data indicate that the low amounts of endometrial LIF expressed during the proliferative phase are physiological, while higher LIF concentrations appear associated with disturbed endometrial proliferation (345).
11. LIF in ectopic pregnancy. A recent study showed elevated
LIF expression in human fallopian tubes, located ipsi- and
contralateral to ectopic pregnancies (346). Cultured human fallopian
tube epithelial cells showed a high constitutive LIF expression and
secretion, while LIF expression in stromal cells was significantly
induced by the inflammatory cytokines IL-1 and TNF (346).
Similarly, in cultured bovine oviduct cells, LIF expression is
stimulated by TNF (347). These findings might provide a link between
salpingitis and ectopic implantation, caused by higher tubal LIF
production, thus providing a more favorable milieu for ectopic
implantation.
12. Summary. In summary, LIF has been demonstrated in several knockout models to be essential for successful murine implantation. In humans, available data also indicate an important auto-/paracrine role for LIF in implantation. Endometrial LIF expression is regulated during the menstrual cycle, peaking in the postovulatory/secretory phase. Some women with previously unexplained infertility show reduced endometrial LIF expression. Further studies are required to examine the incidence and role of disturbed LIF expression in unexplained infertility. The underlying pathophysiology of disturbed endometrial LIF expression or LIF action require further characterization and should provide new therapeutic strategies in the treatment of previously unexplained infertility. Auto/paracrine LIF actions on the preimplantation embryo suggest that there might also be a therapeutic use for LIF in IVF procedures.
B. Hypothalamo-pituitary-adrenal axis
Much insight has recently been gained regarding the
neuro-immuno-endocrine interface involving different cytokines and
pituitary function. Cytokines are secreted as auto/paracrine factors
from pituitary cells and are involved in pituitary development, cell
proliferation, and tumor formation, as well as modulation of hormone
secretion. Several recent reviews address various aspects of this topic
(348, 349, 350, 351, 352, 353). As the auto/paracrine role of LIF in pituitary function and
development has recently received increasing attention, a comprehensive
review of our current understanding of LIF in the pituitary follows.
1. LIF in corticotroph function.
a. LIF and LIF-R expression.
LIF was first described in bovine
pituitary folliculostellate cells by Ferrara et al. (354).
LIF mRNA expression has also been detected in murine corticotroph
AtT-20 cells (47, 355), and pituitary cells derived from mouse (38),
rat (50, 356), and sheep (357). LIF is expressed in human fetal
pituitary as early as 14 weeks of gestation (355), while LIFR and gp130
are expressed in human fetal pituitaries at weeks 18 and 31 of
gestation, as well as in adult pituitary tissue (358) (Fig. 3).
Immunohistochemistry and ligand immunostaining of human fetal pituitary
cells exhibited LIF and LIF binding sites (LIFR) on one third of
ACTH-positive cells and approximately 20% of GH-positive cells,
respectively (355). Ten to 15% of cells that costained with TSH, PRL,
gonadotropins, or -subunit, as well as cells exhibiting no hormone
costaining, were positive for LIF or LIFR, respectively (355). LIF
immunostaining was also detected in all adult human pituitaries
examined and in pituitary somatotroph and corticotroph adenomas (355).
In contrast, in sheep pituitary, LIF immunostaining was found
predominantly in LH- and TSH-positive cells (357). Immunoelectron
microscopy of human fetal pituitary cells (20 weeks gestation) showed
immunostaining with LIF antiserum in the membrane-proximal
cytoplasmic region of secretory granules (355). When cultured,
FACS-sorted LIF-binding human fetal pituicytes showed abundant
ACTH secretion, indicating enrichment of corticotroph cells in this
population (355).
|
In murine hypothalamus and pituitary, LIF gene expression is
up-regulated by lipopolysaccharide (38) or IL-1ß (47), respectively.
Using RT-PCR, both diffusible LIF and matrix-associated LIF and LIFR
were shown to be increased 30 and 60 min after systemic
lipopolysaccharide (LPS) administration (50 µg ip) in murine
hypothalamus and pituitary (38). Northern blot analysis also
demonstrated a severalfold increase of pituitary LIF mRNA 60 min after
systemic administration of IL-1ß (100 ng ip) in C57BL/6 mice (47).
In vitro, incubation of corticotroph AtT-20 cells with
IL-1ß (0.1–10.0 ng/ml) stimulates LIF mRNA expression 5- to 10-fold
(47). This effect can be blocked by coincubation with human IL-1
receptor antagonist (100 ng/ml) or neutralizing mIL-1ß antibody (47).
While TNF (20 ng/ml) exhibits only a modest stimulatory effect on
corticotroph LIF expression in vitro, coincubation of
IL-1ß plus TNF resulted in synergistic induction of LIF expression
in comparison to IL-1ß alone (47).
b. LIF-induced POMC and ACTH.
In vitro, LIF
stimulates ACTH secretion in murine corticotroph AtT-20 cells (163, 206, 355, 360), primary murine (361), rat (356), and ovine (357)
pituitary cultures, as well as in fetal human pituitary cells derived
from week 16–31 gestation (355, 358). Murine corticotroph AtT-20 cells
exhibit a 2- to 4-fold increase of ACTH secretion during incubation
with 1 nM LIF for 24 h (163, 206, 355, 360). While CRH
(10 or 20 nM) alone exerts a 3- to 7-fold stimulation of
ACTH secretion, coincubation of CRH with LIF results in a further
synergistic 2- to 3-fold increase of ACTH secretion in comparison to
CRH alone (163, 355). Similarly, stably transfected AtT-20 cells
overexpressing LIF exhibit a positive correlation between secretion of
LIF and ACTH in conditioned medium and demonstrate enhanced sensitivity
to CRH stimulation, with an increased ACTH production within 8 h
(360). In human fetal pituitary cells, incubation with 1 nM
LIF for 24 h caused a 29% induction of ACTH secretion. CRH (10
nM) alone induced a 3- to 4-fold increase of ACTH
secretion, while coincubation of CRH plus LIF resulted in synergistic
5- to 6-fold elevated ACTH levels (358). Shorter in vitro
incubation with LIF for 6 h had no effect on ACTH levels (358).
LIF-induced ACTH secretion in AtT-20 cells is inhibited by coincubation
with LIFR or gp130 antiserum (163). Coincubation with gp130 antiserum
also decreases LIF-induced ACTH secretion in human fetal pituitary
cells (358). These data demonstrate a specific action of LIF mediated
through its high affinity LIFR-gp130 complex. Coincubation with
specific antisera directed against LIF (355), LIFR (163), or gp130
(358) also decreased basal ACTH secretion rates, demonstrating an
auto/paracrine stimulation of ACTH secretion by corticotroph-derived
LIF. Dexamethasone down-regulates LIF expression in cultured rat
anterior pituitary tissue (50) and inhibits basal and LIF-induced
ACTH secretion (163). Part of the suppressive effect of dexamethasone
on basal ACTH secretion might occur indirectly by suppression of
auto-/paracrine LIF. In favor of this hypothesis, stable overexpression
of LIF in AtT-20 cells blunted dexamethasone suppression of CRH-induced
ACTH secretion (360).
In vivo, systemic LIF administration rapidly induces ACTH
secretion in mice (362) and nonhuman primates (363). In C57BL/6 mice,
ip injection of 12 µg recombinant murine LIF resulted in an
approximately 4-fold increase of ACTH and corticosterone levels at 60
min (362) (Fig. 3). In chronically catheterized fetal rhesus monkeys
(Macaque mulatta), systemic intracarotid administration of
recombinant human LIF (100 µg/kg) was followed by a 12-fold increase
of plasma ACTH levels after 60 min (363). While CRH alone (10 µg/kg)
induced ACTH secretion only 4.8-fold, coadministration of LIF (50
µg/kg) and CRH (10 µg/kg) synergistically stimulated ACTH levels
23-fold in comparison to controls (363).
Stimulation of pituitary ACTH secretion is a characteristic of several gp130-mediated cytokines. In addition to the potent action of LIF, direct stimulation of ACTH secretion in vitro has been demonstrated for OSM (355, 358), IL-11 (364), and IL-6 (355). In mice in vivo, coadministration of IL-1 with each respective member of the IL-6 cytokine family (LIF, OSM, CNTF, IL-11, IL-6, CT-1, NNT-1/BSF-3) induced corticosterone secretion significantly more than IL-1 alone (104, 365).
In addition to ACTH secretion, LIF also stimulates POMC gene expression (163, 177, 206, 355, 364, 366). Incubation of AtT-20 cells with LIF for up to 48 h stimulates POMC mRNA expression about 2-fold (163, 177, 206, 355, 364, 366). In vivo, pituitary POMC mRNA is stimulated in mice 1 and 3 h after systemic LIF administration (362). In AtT-20 cells transfected with a -706/+64 rat POMC promoter-luciferase construct, luc activity was stimulated 2- to 4-fold by LIF (1 nM) alone (163, 177, 206, 364, 366) and up to 7-fold by CRH (10 nM) or (Bu)2cAMP (5 mM) (163, 206, 364, 366). A striking synergism of luc activity was seen during coincubation of CRH and LIF (163, 366). In primary human fetal pituitary cell cultures, a -879/+6 human POMC promoter-luciferase construct was induced 7-fold by LIF (1 nM), 3- to 4-fold by CRH (10 nM), and with potent synergism (22-fold) by CRH plus LIF (358).
The signaling mechanism for LIF, inducing POMC promoter activity, gene expression, and ACTH secretion, has been extensively studied (163, 164, 177, 178, 206, 355, 358, 366).
Immunoneutralization studies show dependence of corticotroph LIF
signaling on LIFR and gp130, indicating specific LIF signaling through
the high-affinity LIFR-gp130 complex (163, 164, 355, 358). In the
corticotroph cell, LIF rapidly induces phosphorylation of gp130 (206),
STAT3 (163, 164, 206), STAT1 (163, 164), STAT1ß (163, 164), and a
novel STAT1-related protein p115 (164). LIF-induced POMC gene
expression and ACTH secretion have recently been shown to be STAT3
dependent in AtT-20 cells (177). Stable transfection of AtT-20 cells
with dominant negative STAT-3 mutants, including mutation of a
carboxy-terminal tyrosine phosphorylation site
Tyr705 to Phe705 (STAT-3F)
or alanine substitutions at positions (E434 and E435) important for DNA
binding (STAT-3D), inhibits LIF-induced POMC gene expression and ACTH
secretion (177). The suppressor of cytokine signaling SOCS-3, harboring
an essential STAT1/STAT3 binding element in its promoter region (178),
is potently stimulated by LIF in AtT-20 cells (178, 206).
Overexpression of SOCS-3, known to inhibit the Jak-STAT signaling
cascade, blocks LIF-induced gp130 and STAT3 phosphorylation in AtT-20
cells, subsequently inhibiting LIF-stimulated POMC promoter activity,
gene expression, and ACTH secretion (206). All these observations
demonstrate pituitary corticotroph LIF signaling through the Jak-STAT
signaling cascade, while alternative LIF signaling pathways,
e.g., MAPK pathway, do not seem to be important in the
corticotroph cell (177).
LIF stimulates promoter activity of a -706/+64 rat POMC
promoter-luciferase construct, alone and in striking synergism with CRH
(163, 366). CRH stimulates POMC expression through a cAMP-dependent
pathway with activation of protein kinase A, associated with increased
intracellular cAMP levels, CREB phosphorylation, and a transient
increase of c-fos mRNA (366). Although LIF stimulates POMC
expression synergistically with CRH, incubation of AtT-20 cells with
LIF does not alter cAMP levels, CREB phosphorylation, or
c-fos mRNA expression (366), indicating a
c-fos-independent pathway for LIF action on CRH-induced POMC
transcription. A -173/-160 element within the rat POMC promoter was
shown, at least in part, to mediate the LIF-CRH synergy on POMC
transcription (366). The nuclear binding factors involved have been
characterized to be serine-phosphorylated proteins (366). In contrast,
STAT3 and STAT1 are apparently not involved in the observed LIF-CRH
synergy on element -173/-160, as no supershift was observed in EMSA
with respective STAT3, STAT1, or antiphosphotyrosine antibodies to
this DNA motif (366). Although LIF affects POMC expression and ACTH
secretion in a STAT-3 dependent manner (177), the specific binding
element in the POMC promoter responsible for this STAT-mediated
activation has not yet been defined.
c. LIF-stress response of the HPA axis.
As discussed
above, in response to various inflammatory stimuli, LIF and LIFR
expression is up-regulated in different tissues. In the circulation,
increased serum levels of LIF have been found in septic patients (361, 367, 368, 369, 370). LIF serum levels are also increased (range, 0.55 to 1.45
ng/ml) in a variety of other acute and chronic inflammatory conditions
(361). Systemic LIF appears to play an essential role in sepsis, as
indicated by observations that serum LIF levels positively correlate
with lethality in sepsis (367, 369, 371). Furthermore, when given a
lethal dose of LPS, preadministration of LIF significantly improved
survival in the mouse (372, 373). Increasing observations suggest an
important role for LIF in the neuro-immuno-endocrine interface,
modulating the pituitary ACTH response to various stimuli, including
inflammatory stimuli and different stressors (38, 47, 362, 374).
LPS injection results in a concordant increase of plasma LIF and ACTH levels in mice (361). This systemic source of LIF might contribute to HPA axis activation in inflammatory states, as systemic LIF injection stimulates the HPA axis in vivo (362, 363). In addition, local expression of murine LIF and LIFR in the hypothalamus and pituitary is up-regulated by systemic LPS administration in vivo (38). However, in vitro, no stimulation of LIF secretion was observed in primary cultured murine pituitary cells during 24 h of LPS incubation (361). This result might be explained by the in vitro system used, possibly lacking other pituitary-derived cells, which might express the essential receptor components for LPS signaling, e.g., CD14 and Toll receptor 4 (375, 376).
IL-1 is an important inflammatory cytokine, thought to activate the HPA axis predominantly through stimulation of hypothalamic CRH and subsequent ACTH secretion (348, 349). IL-1ß stimulates pituitary LIF expression in vivo and in vitro (47). IL-1ß-stimulated ACTH secretion from AtT-20 cells is partially inhibited by coincubation with LIF antiserum (47). After systemic IL-1ß administration in vivo (100 ng ip), LIF- /LIF- knockout mice exhibit reduced ACTH and corticosterone levels in comparison with B6D2F1 wt mice (47). Thus, LIF modulates IL-1ß-induced activation of the HPA axis.
The model of LIF-/LIF- knockout mice has yielded interesting insights into the integrative function of LIF in pituitary function. Baseline plasma ACTH and corticosterone levels are not significantly different (362) or are decreased (47, 374) in LIF-/LIF- knockout in comparison to wt LIF+/LIF+ B6D2F1 mice. As LIF- /LIF- mice show a diminished ACTH response to various stressors (47, 362, 374), stress caused during blood collection, handling of animals, anesthesia, or retroorbital sinus puncture might be responsible for the different baseline ACTH results observed. A small study showed that LIF-/LIF- mice (n = 4–5) exhibited 27% lower baseline ACTH levels and 62% lower "stressed" ACTH levels after 36 h fasting (374). Infusion of LIF-/LIF- mice with recombinant LIF for 3 days (1.2 µg/day) stimulated ACTH and corticosterone levels by 70% and 54%, respectively (374). In a larger study, no significantly altered baseline ACTH and corticosterone levels were found in LIF-/LIF- mice (n = 7–9) (362). After short immobilization stress for 15 min LIF-/LIF- mice exhibited modestly decreased ACTH secretion in comparison with LIF+/LIF+ mice. In contrast, after prolonged immobilization stress for 30 or 45 min, ACTH responses in LIF-/LIF- mice were not different from baseline controls, while LIF+/LIF+ mice demonstrated robustly enhanced ACTH levels (362). Basal and poststress pituitary POMC mRNA content was significantly decreased in LIF-/LIF- mice in comparison with LIF+/LIF+ mice (362). Although LIF-/LIF- mice mounted an attenuated ACTH response, especially to prolonged stressors, serum corticosterone levels after immobilization stress were unaltered in comparison to wt animals (362). This phenomenon can be explained by very small amounts of ACTH being sufficient for a maximal adrenal response.
2. LIF and pituitary development. In murine corticotroph AtT-20 cells, LIF inhibits cell cycle progression from G1 into S phase and proliferation, while enhancing ACTH secretion (377). Thus, LIF acts as a differentiation factor in murine AtT-20 cells, causing a phenotypic switch from proliferative to synthetic (377).
Two transgenic mouse models with pituitary-directed LIF expression have
demonstrated LIF to be an important neuro-immuno-endocrine modulator of
pituitary development (378, 379). Transgenic mice expressing
pituitary-directed LIF driven by the rat GH promoter (378) showed
striking dwarfism, with undetectable serum GH levels and IGF-I levels
diminished to 30% of wt controls. In the pituitary, the number of GH
and PRL cells was decreased, while the number of ACTH cells was
increased 2.2-fold. The anterior pituitary contained cystic cavities,
lined by cuboidal, ciliated epithelial cells, focally immunopositive
for cytokeratin and S-100 protein and immunonegative for
adenohypophyseal hormones. Human Rathke’s cysts also exhibit LIF
immunoreactivity in cyst-lining cells. Thus, LIF overexpression might
perturb differentiation of Rathke’s pouch, an invagination of oral
ectoderm and source of common progenitor cells, believed to
differentiate into distinct hormone-secreting cell lines (378). During
murine pituitary ontogeny, GH expression emerges at a relatively late
stage [embryonic day 16–17 (E16–17)], while GSU is the
earliest hormone-specific transcript (E9.5), followed by POMC (E12),
TSH (E12–13), and LH and FSH (E15). To examine the impact of LIF on
earlier pituitary development, a second transgenic model with
pituitarydirected LIF driven by the GSU promoter was
established (379). Phenotypically, these transgenic mice were dwarfs
with extremely low IGF-I serum levels. Infertility of both sexes was
due to central hypogonadotropism. In comparison to wt mice, the
transgenic mice exhibited Cushingoid features including truncal obesity
and thin skin, elevated basal corticosterone levels, and incomplete
suppression of corticosterone levels by dexamethasone (379). The LIF
transgenic pituitary was hypoplastic due to a dramatic decrease of
somatotroph, lactotroph, and gonadotroph cells, as well as a variably
diminished number of thyrotroph cells. ACTHimmunopositive cells
were increased in absolute numbers and accounted for approximately 65%
of anterior pituitary cells in the LIF-transgenic pituitaries, in
comparison with about 13% in wt pituitaries (379). On E14.5, the
transcription factors Lhx3 and Pit-1 were also decreased in transgenic
pituitaries (379). As a hypothetical model, suppression of LHX3
expression in the fetal pituitary by LIF might direct differentiation
of progenitor cells away from Lhx3-dependent cell lineages
(gonadotroph, thyrotroph, somatotroph, and lactotroph) toward the
corticotroph lineage and ciliated epithelial cells (379) (Fig. 4).
|
C. Bone metabolism
Several cytokines play a role in proliferation and
function of osteoblasts and osteoclasts, thus affecting physiological
bone formation and remodeling, as well as pathophysiological states,
e.g., osteoporosis. Several current reviews (380, 381, 382, 383, 384, 385)
discuss the growing evidence supporting the role of gp130-sharing
cytokines, especially IL-6 and IL-11, in osteoclast differentiation and
bone resorption. IL-6 and IL-11 are secreted by osteoblast-like cells
and act in a paracrine fashion on osteoclasts. IL-6 expression in
osteoblast-like cells is repressed by estrogens (385) and androgens
(386), and stimulated by T3 (387, 388, 389, 390). Loss of estrogens
also increases IL-6R and gp130 expression in osteoblasts (388). Thus,
lowering sex steroids or hyperthyroidism might be associated with
increased bone resorption and increased risk of osteoporosis due to
increased osteoblast IL-6 expression, subsequently stimulating
osteoclast differentiation and proliferation.
In addition, other cytokines of the IL-6 family, namely LIF and OSM, exert effects on bone resorption and formation. Although the topic of "LIF and bone metabolism" has been extensively reviewed several years ago (391, 392), recent studies have provided new insights for specific LIF function in bone metabolism. Similar to other cytokine functions, these effects of members of the IL-6 cytokine family on bone metabolism appear to partially overlap.
1. LIF expression on osteoblasts and osteoclasts. LIF
expression has been reported in various osteoblastic cell lines,
including murine MC3T3-E1 (393, 394, 395), rat UMR 106–06 (396), rat UMR
201 (396), in primary cell cultures derived from newborn rat long bones
(396) or fetal rat calvarias (397), and finally in U-OS and SaoS-2
human osteosarcoma cell lines (398), as well as in benign and malignant
human primary bone tumors (399). As demonstrated by Northern blot
analysis, LIF expression in murine MC3T3-E1 cells is rapidly and
transiently stimulated by IL-1, IL-1ß, TNF-, or LPS (395).
Similarly, LIF expression in rat UMR 201 cells is stimulated by TNF
(396). By Northern blot analysis, no stimulation of LIF mRNA expression
in MC3T3-E1 cells was observed after incubation with PTH,
1,25(OH)2D3, or LIF
itself (395). However, using more sensitive semiquantitative RT-PCR
analysis, a rapid and transient stimulation of LIF and IL-6 mRNA was
detected in MC3T3-E1 cells incubated with PTH (394). Stimulation of LIF
and IL-6 expression by PTH in osteoblasts is an immediate-early gene
response induced by cAMP signal transduction (400). Also in
vivo, injection of PTH into the subcutaneous space overlying mouse
parietal bones rapidly and transiently induces parietal expression of
LIF and IL-6 transcripts (401), suggesting that LIF and IL-6 may be
mediators of initial PTH effects in vivo.
2. LIFR expression on osteoblasts and osteoclasts. Using ligand autoradiography, specific LIF binding was found in osteoblasts, but not in multinucleated osteoclasts derived from newborn rat long bones (396). Scatchard analysis of the osteoblast-like rat osteogenic sarcoma cell line UMR 106–06 revealed 300 LIF binding sites per cell with a Kd of 60 pM (396). A similar number and affinity for LIF receptors was found on preosteoblastic rat calvaria (RCT-1) cells (402). By RT-PCR, expression of gp130, LIFR, and IL-6R has been demonstrated in primary calvarial cultures isolated from 21-day-old rat fetuses (397).
Murine osteoblastic MC3T3-E1 cells exhibit approximately 1,100 LIF binding sites per cell with a Kd of 161 pM (394). Immunoprecipitation verified expression of LIFR and an alternative gp130 form in MC3T3-E1 cells (153, 390). LIFinduced signal transduction in osteoblastic MC3T3-E1 cells involves tyrosine phosphorylation of Jak1 and, to a lesser extent, Jak2 (153), gp130 (403), and LIFR (153, 403), as well as predominantly STAT1 and, to a lesser extent, STAT3 (153). LIF stimulates expression of gp130 in MC3T3-E1 cells (390).
In contrast, human osteoblast-like MG-63 osteosarcoma cells do not express LIFRs (403, 404). LIFR antibodies failed to precipitate a specific protein (403), and LIF does not phosphorylate gp130 (403), form STAT1 and STAT3 complexes (404), or activate MAPK (404) in these cells. However, despite the lack of LIF activity in MG-63 cells, OSM stimulates gp130 tyrosine phosphorylation (403), STAT1, and STAT3 (404), as well as Erk1 and Erk2 (376), probably acting through a specific OSMR type II (403).
Both murine MC3T3-E1 and human MG-63 cells are modestly induced by IL-6 plus sIL-6R and IL-11, respectively (403). Thus, various members of the IL-6 cytokine family can affect osteoblastic MC3T3-E1 or MG-63 cells. Depending on the cell type-specific expression of LIFR or OSMR type II, LIF and OSM appear to exhibit overlapping effects mediated by shared LIFR signaling, or selective effects of OSM, signaling through the specific OSMR type II.
3. LIF effect on bone resorption and bone formation. LIF was first reported to stimulate bone resorption by Abe et al. (405), when LIF purified from conditioned medium of mitogen-activated spleen cells exhibited osteoclast-activating factor activity. In vitro, LIF induced bone resorption in neonatal mouse calvaria cell cultures and increased osteoclast numbers (406). But, in the same culture system, LIF also induced 3H-thymidine incorporation in AP-positive stained osteoblastic cells and in the osteoprogenitor region of cultured hemicalvaria of 6-day-old mice (406, 407). In fetal mouse calvarial cultures, a combination of LIF plus IL-1 induced bone resorption, while each cytokine by itself was inactive (395). In vivo, local injection of LIF (0.5 µg/day) over a hemicalvarial region for 5 days accelerated bone turnover (408). LIF increased bone resorption as evidenced by increased osteoblast numbers (3-fold), osteoclast surface (5-fold), and eroded bone surface (10-fold) (408). However, net bone formation was also increased, as osteoblast numbers, osteoblast surface, osteoid area, and overall bone thickness doubled (408). Systemic overexpression of LIF in syngeneic DBA/2 mice engrafted with hemopoietic FDC-P1 cells transfected with LIF cDNA resulted in increased osteoclastic resorption, as well as increased numbers of osteoblasts and a large increase in net bone formation and bone mass (409). Thus available data suggest a stimulatory effect of LIF on bone resorption by inducing osteoclast proliferation and differentiation (395, 405, 406, 410), while few studies report an inhibitory effect of LIF on bone resorption (411, 412). Another study reported LIF to increase osteoclastic activity in cultured cells derived from a giant cell tumor of bone, while osteoclast numbers did not change significantly (413).
Although most available data suggest a stimulatory effect of IL-6 family cytokines on bone resorption by inducing osteoclast proliferation and differentiation (380, 381, 382, 383, 384, 385, 395, 405, 406, 410), osteoclasts are not dependent on gp130 signaling, as osteoclasts are present in gp130-deficient mice (414). LIFR-/- knockout mice exhibit bone development abnormalities with an approximately two thirds reduction in bone mass, 6-fold increase in osteoclast numbers, and 7-fold increase in osteoclast surfaces, while bone formation is only nonsignificantly reduced by 30% (249). In view of the indirect osteoclast stimulation by LIF (395, 405, 406, 410), these findings (249) are unexpected. Other indirect effects of LIF causing a balance between osteoclast activation and inhibition might contribute to this finding. Similarly, other cytokines using the LIFR for signaling might exert negative direct or indirect effects on osteoclast proliferation and function.
Bone remodeling is a dynamic process of osteoclastmediated bone
resorption and subsequent bone formation by osteoblasts. A mechanism
for osteoblasts modulating proliferation and differentiation of
osteoclast progenitor cells is by secretion of cytokines, which
stimulate osteoclasts in a paracrine fashion (380, 381, 382, 383, 384, 385). Stimulation of
osteoblasts with IL-1, TNF, or PTH induces expression of IL-6
(380, 381, 382, 383, 384, 385), IL-11 (415, 416), and LIF (38, 397, 401), respectively. A
direct paracrine effect of osteoblast-derived LIF on osteoclasts is
unlikely, as LIFRs have not been demonstrated on osteoclasts, but only
on osteoblasts (396). However, LIF stimulates IL-6 expression from
fetal rat calvaria cell cultures (397) and neonatal mouse osteoblasts
(417). Thus, mitogenic LIF effects on osteoclasts could be indirect,
mediated by osteoblast-derived IL-6. This theory of LIF acting
indirectly via osteoblasts on osteoclasts is also supported by the
observation that LIF stimulates bone resorption only in cultures
containing both osteoclasts and osteoblasts, while highly purified
osteoclast cultures were not stimulated by LIF (392). In addition, as
LIF stimulates collagenase-3 expression in osteoblasts, the increase in
collagenase might be responsible, in part, for the stimulation of
osteolytic activity by LIF (418). On the other hand, although
osteoclast-like cells have been considered to lack LIFR expression
(396), osteoclast-like cells from a human giant cell tumor of the bone
have been found to express LIF and LIFR (419). In these multinucleated
giant cells, LIF directly stimulated proliferation, although decreasing
their ability of resorption (419). Therefore, at least in this tumor
cell model, direct autocrine/paracrine LIF effects in osteoclast-like
cells seem to exist.
Conflicting effects of LIF on differentiation and proliferation of osteoblast-like cells have been reported (402, 420). Osteoblast differentiation from murine embryonic fibroblasts is not stimulated by LIF, OSM, or CNTF, while IL-6 plus sIL-6R, or IL-11 promote differentiation of AP-positive cells (421). Accordingly, murine embryonic fibroblasts express gp130, but not the IL-6R(gp80) or the LIFR (gp190) (421). LIF was observed to either inhibit DNA synthesis in MC3T3-E1 cells (153, 420) or to have no effect (422), while OSM inhibited 3H-thymidine incorporation in MG-63 cells (404). The prodifferentiation and antiapoptotic effects of OSM in MG-63 cells may be mediated by the cyclin-dependent kinase inhibitor p21 (423). Similarly, DNA synthesis in calvarial osteoblast cultures derived from newborn mice (153) and bone nodule formation in primary cultures of rat calvarial cells derived from 21-day old fetuses (397) were inhibited by LIF. In contrast, high concentrations of LIF (10 to 1,000 ng/ml) increased DNA synthesis and 3H-thymidine incorporation of AP-positive cells in human trabecular bone cultures (424). LIF (100 ng/ml) and OSM (100 ng/ml) stimulated 3H-thymidine incorporation 2.5- to 3.0-fold in primary cultures of parietal bone-derived osteoblastic cultures isolated from 22-day old rat fetuses (417). LIF also stimulated in vitro proliferation of stromal progenitors with osteogenic potential from isolated bone marrow (424). In a murine heterotopic calcification model, LIF and OSM lowered the Ca/P ratio and mineral density of newly induced ossifications (425). Therefore, in addition to an increase of net bone formation, LIF also seems to alter the mineral phase quality of the newly formed bone.
4. Summary. In summary, LIF modulates bone formation by direct
or indirect paracrine effects on osteoblasts and osteoclasts,
respectively. Several cytokines, including IL-1, TNF, LPS, and PTH
stimulate LIF secretion from osteoblastic cells. LIF is believed to act
indirectly on osteoclasts by stimulating stromal/osteoblastic
expression of other cytokines, which mediate LIF-induced osteoclast
proliferation and bone resorption (Fig. 5). In addition to indirect stimulation
of bone resorption, LIF also increases new bone formation, resulting in
increased net bone mass. The observed contradictory results of LIF on
osteoblasts and osteoclasts might be explained by several factors.
First, different experimental conditions, e.g., cell
isolation procedures, might per se influence cell subsets
and their specific responsiveness. Second, as cell preparations for
primary culture are isolated from animals of different ages,
osteoblasts at different stages of development might respond
differently to LIF. Future studies should focus on experiments
evaluating the specific and essential role of LIF and mechanisms for
direct or indirect osteoclast stimulation.
|
, IL-6,
LIF, IFN, and others in inducing weight loss (426, 427, 428).
Based on studies from Mori et al. (429, 430) and others
(257, 431, 432, 433, 434), LIF is thought to play a role in the cancer cachexia
syndrome by inhibition of adipocyte lipoprotein lipase (LPL) activity.
Similarly, IL-1, TNF, IL-6, and IFN decrease LPL activity (for
review see Ref. 433). In rats, systemic LIF administration increases
hepatic triglyceride secretion by stimulating both lipolysis and
de novo fatty acid synthesis (435).
Serum levels of LIF are increased in most patients with cancer and
lymphomas (361, 399, 436). Nude mice bearing tumors of the SEKI human
melanoma cell line develop cachexia and loose 25–40% of total body
weight (429, 432). Resection of the tumor results in normalization of
body weight and abrogation of the cachexia (429). Mori et
al. purified a "lipoprotein lipase-inhibitor" derived from
conditioned media of SEKI human melanoma cells, and subsequently found
it to be identical with LIF (429, 430). Injection of six different
human carcinoma cell lines in nude mice resulted in cachexia and
substantial weight loss in each case (431, 432). However, while SEKI
(melanoma), NAGAI (neuroepithelioma), and OCC-1C (oral cavity
carcinoma) cells were associated with high levels of LIF expression and
secretion, MKN-1 (gastric carcinoma), LS180 (colon carcinoma), or LX-1
(lung carcinoma) cell lines do not express LIF, IL-6, or IL-11 (432).
Accordingly, conditioned medium derived from SEKI, NAGAI, or OCC-1C
cultures inhibits LPL activity by 80–90%, while conditioned medium
derived from MKN-1, LS180, or LX-1 cultures causes only a 20–30%
decrease of LPL activity (432). These data indicate that some tumor
cells cause cachexia by production of LIF and related cytokines and
subsequent inhibition of LPL activity. However, other tumors may cause
similar cancer cachexia syndromes by other, LIF-independent mechanisms
(432). LIF signaling in 3T3-L1 adipocytes involves tyrosine
phosphorylation of STAT 3 and STAT1 (155, 167). LPL activity in 3T3-L1
cells is decreased by LIF, mostly by inhibition of LPL transcription
(433). Although showing similar half-maximal doses for inhibition of
LPL activity in 3T3-L1 cells, LIF decreased LL activity less potently
than TNF (433). Additional proof supporting a role for LIF in energy
homeostasis derives from the observation that mice engrafted with cells
producing high systemic LIF levels developed a fatal syndrome
accompanied by weight loss of 20–25%, associated with loss of all
subcutaneous and abdominal fat and generalized organ atrophy within
12–70 days (409). Similarly, injection of recombinant human LIF
to nonhuman primates (80 µg/kg/day for 14 consecutive days) results
in weight loss of 10% and a reduction in subcutaneous fatty tissue
(257).
The cytokine-induced decrease in adipocyte LPL expression and decreased
LPL activity is a possible mechanism for LIF causing cachexia. However,
anorexia, which often accompanies infectious diseases or cancer, cannot
be explained by this mechanism. In a recent study by Sarraf et
al. (427), LPS, TNF, IL-1, and to a somewhat lesser degree LIF
and IL-6, were found to stimulate murine leptin mRNA expression in fat
and serum leptin levels. Thus, the anorectic effect of inflammatory
cytokines might be mediated, at least in part, by leptin, and in
cancer-associated anorexia, cytokine-mediated regulation of adipocyte
leptin might be involved. However, direct expression of leptin could
not be detected by RT-PCR in several human cancer cell lines (432).
1. Summary. In summary, LIF induces weight loss and cachexia
in vivo. Two possible mechanisms might mediate this
LIF-induced weight loss including LIF-induced decrease in adipocyte LPL
activity, and LIF-induced increase of serum leptin levels. However,
these effects are not specific LIF actions. Similar effects can also be
caused by other cytokines, e.g., IL-1 and TNF-. As many
tumor patients show elevated LIF serum levels and various tumor cell
lines secrete LIF, LIF is one potential cachectic factor in these
patients.
E. Endocrine-responsive tumors
In 90% of cancer patients (n = 75) LIF was detectable in the
serum with a median value of 0.4 ± 0.1ng/ml (361). No differences
in LIF levels were observed in patients with different carcinoma types
(361). Significantly increased LIF serum levels were also found in
Hodgkin’s and Non-Hodgkin lymphomas in comparison to controls (436).
In vitro, human carcinoma cell lines derived from pancreas
(n = 8), lung (n = 7), stomach (n = 5), colon (n =
2), breast (n = 2), melanocytes (n = 2), liver (n = 1),
and gall bladder (n = 1), all exhibited LIF mRNA expression
detected by Northern blot analysis (437, 438). gp130 And LIFR were also
detected by RT-PCR in virtually all examined cancer cell lines (438).
Thus, LIF, LIFR, and gp130 seem to be constitutively expressed in most
cancer cells, indicating the possibility of auto/paracrine stimulation
of cancer cells by LIF. Secretion of either LIF or IL-6 from the murine
sarcoma cell line 4JK is induced by IL-1 and TNF severalfold (439).
Culture of RAW 264 macrophages with conditioned medium from 4JK cells
activates TNF secretion about 10-fold, while coculture of RAW and
4JK cells results in increased LIF and IL-6 production (439). These
results suggest an interaction of tumor-infiltrating
monocytes/macrophages and sarcoma cells, to enhance tumor cell LIF
secretion. Both induction of cell proliferation as well as growth
inhibition and apoptosis can be stimulated by LIF (438), indicating
cell type-specific signal transduction in different cancer cells.
LIFR and gp130 are expressed in many human breast carcinoma cell lines (440), as well as human breast cancer specimens (440, 441) and human mammary epithelial cells derived from normal breast tissue (441, 442). LIFR and gp130 were detected by RT-PCR in human breast cell lines, including 184 and 184B5 cells derived from nonmalignant breast epithelial cells; MCF-7M, T-47D, MDA-MB-134, and MDA-MB-361cells derived from estrogen receptor-positive breast cancer cells; and ASK-BR-3, BT-20, BT-549, and MDA-MB-231 derived from estrogen receptor-negative breast cancer cells, respectively (440). Similarly, IL-11R was also expressed, while transcripts for the IL-6R and CNTFR were not detected in the examined cell lines (440). Ligand binding of 125I-LIF to MCF-7M cells and Scatchard plot analysis revealed a single class of high-affinity (Kd 1.5 x 10-11 M to 27.0 x 10-11 M) binding sites (60–430 receptors per cell) (440, 443).
In most studies, LIF has been reported to stimulate proliferation of estrogen receptor-positive MCF-7M cells (438, 442, 443, 444) and T-47D cells (443, 444). MCF-7M cells (443, 444) and T-47D cells (444, 445, 446) do not express LIF, indicating a paracrine role of LIF, possibly derived from adjacent nontumorous cells. LIF also stimulated proliferation of the estrogen receptor-negative breast tumor cells SK-BR3 (442, 443) and BT20 (442). However, LIF showed no effect on proliferation of estrogen receptor-negative breast tumor cells BT-549 and MDA-MB-231 (440), as well as normal human breast cells, including primary human mammary epithelial cells, HBL 1000 and HS578BST (442, 443). Despite being nonresponsive to LIF (444), estrogen receptor-negative MDA-MB-231 cells express and secrete LIF (444, 446). A 666-nucleotide human LIF promoter luciferase construct, cotransfected together with progesterone receptors into MDA-MB-231 cells, showed stimulated luc activity after stimulation with medroxy-progesterone acetate (446). LIF treatment of primary human breast cancer cells, derived from 6 patients, resulted in a 12–110% increase in colony formation (443). In contrast to these data indicating a stimulatory effect of LIF on proliferation of different breast cancer cells (438, 442, 443, 444), one study observed an inhibitory effect of LIF on proliferation of MCF-7M cells (440). This discrepancy might be due to different batches of MCF-7M cells exhibiting different states of differentiation, as well as different incubation time periods.
OSM inhibits proliferation of MCF-7M, SK-BR3, BT-549, and MDA-MB231 cells, as well as normal human mammary epithelial cells (440, 442). These opposing effects of LIF and OSM on cell proliferation may be explained by differing signal transduction of LIF and OSM through either the LIFR (shared by LIF and OSM, OSMR type I) or the OSMR (specific for OSM, OSMR type II), respectively. OSMR are more abundantly expressed than LIFR in normal human mammary epithelial cells and in most breast cancer cell lines (440, 442).
RT-PCR of mRNA derived from 50 human breast cancer specimens revealed expression of transcripts for gp130, LIFR, IL-11R, IL-6R, and CNTFR in virtually all samples (440). A small study reported detection of LIF, IL-6, IL-11, and OSM transcripts by RT-PCR in breast tumor samples (445). In another study, immunohistochemistry of 50 human breast cancer specimens revealed staining for LIF and LIFR in 80% of the tumors, as well as most adjacent normal breast epithelium (441). In normal breast epithelium, immunostaining for LIF and LIFR did not differ in samples derived from premenopausal and postmenopausal women (441). In breast cancer, LIF and LIFR coexpression correlated significantly with diploidy and a low S-phase fraction (441).
1. Summary. In summary, LIF plays an important, yet incompletely understood, role in breast cancer proliferation. First, LIF is expressed by various cancer cell lines and in primary tumors (441, 444, 445, 446). LIFR and gp130 are expressed in most breast cancer cells (440, 441, 442), and the data suggest a paracrine stimulatory effect of LIF on breast cancer cell proliferation. Second, as LIF indirectly stimulates osteoclast proliferation and bone resorption, LIF derived from breast cancer cells could stimulate osteoclasts in the process of bone metastasis. The mouse mammary tumor cell line MMT060562 secretes LIF and supports osteoclast formation in a mouse bone marrow coculture system (447). In addition, the osteoclast-activating fraction of the conditioned medium derived from MMT060562 cells could be inactivated by LIF antibody (447).
Therefore, the role of LIF in breast cancer might be of clinical relevance and merits further evaluation. Targeted overexpression of inhibitors of cytokine signaling (SOCS proteins) in breast carcinoma cell lines might provide insight in the effect of different gp130 sharing cytokines stimulating these cells and also prove be a potential therapeutic tool to disrupt auto-/paracrine stimulation of tumor growth by LIF and other IL-6 cytokine family members.
|
VII. Integrative Section—The Neuroimmune-Endocrine Interface |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
LIF, in this regard, appears to play a critical role in neuroimmune transduction of central and immune signals to the adrenal axis. Furthermore, LIF regulation of cell growth, reproductive function, bone metabolism, and energy homeostasis all point to a pivotal role for gp-130-mediated signaling in endocrine control. Further understanding of mechanisms regulating LIF expression and LIFR signaling will thus elucidate multiple endocrine-regulatory processes.
Clearly, the regulatory biology of cytokine synthesis and action impacts on protean physiological and pathological processes. Perhaps as a reflection of their ubiquitous yet critical actions, cytokines and their respective signaling molecules share significant structural and functional overlap and redundancy.
|
Footnotes |
|---|
1 This work was supported in part by a scholarship of the Deutsche
Forschungsgemeinschaft (Au 139/1–1) and by NIH grant DK 50238. 
2 Current address: Department of Internal Medicine II, Klinikum
Grosshadern, Ludwig-Maximilians-University, Munich 81366, Germany.
|
References |
|---|
|
TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
|---|
. J Immunol 150:1496–1502[Abstract]
. Arthritis Rheum 36:790–794[Medline]
and glucocorticoids. Blood 86:3763–3770-chain. Expression in
normal, gestating, and leukemia inhibitory factor nullizygous mice.
J Biol Chem 271:5495–5504-chain in normal human
urine and plasma. J Biol Chem 273:10798–10805-chain can act as an IL-11 antagonist. Blood 90:4403–4412 component as a soluble mediator of CNTF responses. Science 259:1736–1739: production, binding
stoichiometry, and characterization of its activity as a diffusible
factor. Biochemistry 33:5813–5818[CrossRef][Medline]
-chain is determined primarily
by the immunoglobulin-like domain. J Biol Chem 272:23976–23985 and stoichiometry of in vitro IL-11 receptor
complexes with gp130. J Biol Chem 271:30986–30991 in
adipocytes. J Biol Chem 273:31408–31416 and gp130 signaling molecules differ in their sequence
preferences and transcriptional induction properties. Nucleic Acids Res 23:3283–3289-inducible gene and confers resistance to interferons.
Blood 92:1668–1676.
J Immunol 156:4401–4407[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  G. Song, M C. Satterfield, J. Kim, F. W Bazer, and T. E Spencer Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy Reproduction, March 1, 2009; 137(3): 553 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. V Mikhaylova, T. Jaaskelainen, J. Jaaskelainen, J. J Palvimo, and R. Voutilainen Leukemia inhibitory factor as a regulator of steroidogenesis in human NCI-H295R adrenocortical cells J. Endocrinol., December 1, 2008; 199(3): 435 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Labeur, D. Refojo, B. Wolfel, J. Stalla, V. Vargas, M. Theodoropoulou, M. Buchfelder, M. Paez-Pereda, E. Arzt, and G. K Stalla Interferon-{gamma} inhibits cellular proliferation and ACTH production in corticotroph tumor cells through a novel janus kinases-signal transducer and activator of transcription 1/nuclear factor-kappa B inhibitory signaling pathway J. Endocrinol., November 1, 2008; 199(2): 177 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamanaka Pluripotency and nuclear reprogramming Phil Trans R Soc B, June 27, 2008; 363(1500): 2079 - 2087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Endoh, T. A. Endo, T. Endoh, Y.-i. Fujimura, O. Ohara, T. Toyoda, A. P. Otte, M. Okano, N. Brockdorff, M. Vidal, et al. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity Development, April 15, 2008; 135(8): 1513 - 1524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. H. Damon TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3 Am J Physiol Cell Physiol, January 1, 2008; 294(1): C306 - C312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Wanggren, P.G. Lalitkumar, F. Hambiliki, B. Stabi, K. Gemzell-Danielsson, and A. Stavreus-Evers Leukaemia inhibitory factor receptor and gp130 in the human Fallopian tube and endometrium before and after mifepristone treatment and in the human preimplantation embryo Mol. Hum. Reprod., June 1, 2007; 13(6): 391 - 397. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-O. Jansson, S. Moverare-Skrtic, A. Berndtsson, I. Wernstedt, H. Carlsten, and C. Ohlsson Leukemia inhibitory factor reduces body fat mass in ovariectomized mice Eur. J. Endocrinol., February 1, 2006; 154(2): 349 - 354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kariagina, S. Zonis, M. Afkhami, D. Romanenko, and V. Chesnokova Leukemia inhibitory factor regulates glucocorticoid receptor expression in the hypothalamic-pituitary-adrenal axis Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E857 - E863. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Catalano, M. H. Johnson, E. A. Campbell, D. S. Charnock-Jones, S. K. Smith, and A. M. Sharkey Inhibition of Stat3 activation in the endometrium prevents implantation: A nonsteroidal approach to contraception PNAS, June 14, 2005; 102(24): 8585 - 8590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-J. Kim, J.-I. Park, and B. D. Nelkin IFI16 Is an Essential Mediator of Growth Inhibition, but Not Differentiation, Induced by the Leukemia Inhibitory Factor/JAK/STAT Pathway in Medullary Thyroid Carcinoma Cells J. Biol. Chem., February 11, 2005; 280(6): 4913 - 4920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Nozaki, J. G. Lunz III, S. Specht, J.-I. Park, A. S. Giraud, N. Murase, and A. J. Demetris Regulation and Function of Trefoil Factor Family 3 Expression in the Biliary Tree Am. J. Pathol., December 1, 2004; 165(6): 1907 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. A. Sherwin, T. C. Freeman, R. J. Stephens, S. Kimber, A. G. Smith, I. Chambers, S. K. Smith, and A. M. Sharkey Identification of Genes Regulated by Leukemia-Inhibitory Factor in the Mouse Uterus at the Time of Implantation Mol. Endocrinol., September 1, 2004; 18(9): 2185 - 2195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Suzuki, L. S. Wright, P. Marwah, H. A. Lardy, and C. N. Svendsen Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex PNAS, March 2, 2004; 101(9): 3202 - 3207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Abbud, R. Kelleher, and S. Melmed Cell-Specific Pituitary Gene Expression Profiles after Treatment with Leukemia Inhibitory Factor Reveal Novel Modulators for Proopiomelanocortin Expression Endocrinology, February 1, 2004; 145(2): 867 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vlotides, K. Zitzmann, S. Hengge, D. Engelhardt, G. K. Stalla, and C. J. Auernhammer Expression of Novel Neurotrophin-1/B-Cell Stimulating Factor-3 (NNT-1/BSF-3) in Murine Pituitary Folliculostellate TtT/GF Cells: Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Peptide-Induced Stimulation of NNT-1/BSF-3 Is Mediated by Protein Kinase A, Protein Kinase C, and Extracellular-Signal-Regulated Kinase1/2 Pathways Endocrinology, February 1, 2004; 145(2): 716 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Nightingale, S. Patel, N. Suzuki, R. Buxton, K.-i. Takagi, J. Suzuki, Y. Sumi, A. Imaizumi, R. M. Mason, and Z. Zhang Oncostatin M, a Cytokine Released by Activated Mononuclear Cells, Induces Epithelial Cell-Myofibroblast Transdifferentiation via Jak/Stat Pathway Activation J. Am. Soc. Nephrol., January 1, 2004; 15(1): 21 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kariagina, D. Romanenko, S.-G. Ren, and V. Chesnokova Hypothalamic-Pituitary Cytokine Network Endocrinology, January 1, 2004; 145(1): 104 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Fedorcsak and R. Storeng Effects of Leptin and Leukemia Inhibitory Factor on Preimplantation Development and STAT3 Signaling of Mouse Embryos In Vitro Biol Reprod, November 1, 2003; 69(5): 1531 - 1538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Auernhammer, N. B. Isele, F. B. Kopp, G. Spoettl, N. Cengic, M. M. Weber, G. Senaldi, and D. Engelhardt Novel Neurotrophin-1/B Cell-Stimulating Factor-3 (Cardiotrophin-Like Cytokine) Stimulates Corticotroph Function via a Signal Transducer and Activator of Transcription-Dependent Mechanism Negatively Regulated by Suppressor of Cytokine Signaling-3 Endocrinology, April 1, 2003; 144(4): 1202 - 1210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Beishuizen and L. G. Thijs Review: Endotoxin and the hypothalamo-pituitary-adrenal (HPA) axis Innate Immunity, February 1, 2003; 9(1): 3 - 24. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Ben-Shlomo, I. Miklovsky, S.-G. Ren, W. H. Yong, A. P. Heaney, M. D. Culler, and S. Melmed Leukemia Inhibitory Factor Regulates Prolactin Secretion in Prolactinoma and Lactotroph Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 858 - 863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Castro, D. Giacomini, A. C. Nagashima, C. Onofri, M. Graciarena, K. Kobayashi, M. Paez-Pereda, U. Renner, G. K. Stalla, and E. Arzt Reduced Expression of the Cytokine Transducer gp130 Inhibits Hormone Secretion, Cell Growth, and Tumor Development of Pituitary Lactosomatotrophic GH3 Cells Endocrinology, February 1, 2003; 144(2): 693 - 700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-I. Park, C. J. Strock, D. W. Ball, and B. D. Nelkin The Ras/Raf/MEK/Extracellular Signal-Regulated Kinase Pathway Induces Autocrine-Paracrine Growth Inhibition via the Leukemia Inhibitory Factor/JAK/STAT Pathway Mol. Cell. Biol., January 15, 2003; 23(2): 543 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Metcalf The Unsolved Enigmas of Leukemia Inhibitory Factor Stem Cells, January 1, 2003; 21(1): 5 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Mynard, L. Guignat, J. Devin-Leclerc, X. Bertagna, and M. G. Catelli Different Mechanisms for Leukemia Inhibitory Factor-Dependent Activation of Two Proopiomelanocortin Promoter Regions Endocrinology, October 1, 2002; 143(10): 3916 - 3924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Toniato, X. P. Chen, J. Losman, V. Flati, L. Donahue, and P. Rothman TRIM8/GERP RING Finger Protein Interacts with SOCS-1 J. Biol. Chem., September 27, 2002; 277(40): 37315 - 37322. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Palmqvist, E. Persson, H. H. Conaway, and U. H. Lerner IL-6, Leukemia Inhibitory Factor, and Oncostatin M Stimulate Bone Resorption and Regulate the Expression of Receptor Activator of NF-{kappa}B Ligand, Osteoprotegerin, and Receptor Activator of NF-{kappa}B in Mouse Calvariae J. Immunol., September 15, 2002; 169(6): 3353 - 3362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E. Spangenburg and F. W. Booth Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation Am J Physiol Cell Physiol, July 1, 2002; 283(1): C204 - C211. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Chesnokova and S. Melmed Minireview: Neuro-Immuno-Endocrine Modulation of the Hypothalamic-Pituitary-Adrenal (HPA) Axis by gp130 Signaling Molecules Endocrinology, May 1, 2002; 143(5): 1571 - 1574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Blanchard, Y. Wang, E. Kinzie, L. Duplomb, A. Godard, and H. Baumann Oncostatin M Regulates the Synthesis and Turnover of gp130, Leukemia Inhibitory Factor Receptor alpha , and Oncostatin M Receptor beta by Distinct Mechanisms J. Biol. Chem., December 7, 2001; 276(50): 47038 - 47045. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bousquet, V. Chesnokova, A. Kariagina, A. Ferrand, and S. Melmed cAMP Neuropeptide Agonists Induce Pituitary Suppressor of Cytokine Signaling-3: Novel Negative Feedback Mechanism for Corticotroph Cytokine Action Mol. Endocrinol., November 1, 2001; 15(11): 1880 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-G. Cheng, J. R. Chen, L. Hernandez, W. G. Alvord, and C. L. Stewart Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation PNAS, June 28, 2001; (2001) 151180898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mauduit, I. Goddard, V. Besset, E. Tabone, C. Rey, F. Gasnier, F. Dacheux, and M. Benahmed Leukemia Inhibitory Factor Antagonizes Gonadotropin Induced-Testosterone Synthesis in Cultured Porcine Leydig Cells: Sites of Action Endocrinology, June 1, 2001; 142(6): 2509 - 2520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Chen, J.-G. Cheng, T. Shatzer, L. Sewell, L. Hernandez, and C. L. Stewart Leukemia Inhibitory Factor Can Substitute for Nidatory Estrogen and Is Essential to Inducing a Receptive Uterus for Implantation But Is Not Essential for Subsequent Embryogenesis Endocrinology, December 1, 2000; 141(12): 4365 - 4372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-G. Cheng, J. R. Chen, L. Hernandez, W. G. Alvord, and C. L. Stewart Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation PNAS, July 17, 2001; 98(15): 8680 - 8685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Chesnokova, A. Kariagina, and S. Melmed Opposing effects of pituitary leukemia inhibitory factor and SOCS-3 on the ACTH axis response to inflammation Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1110 - E1118. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
