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Academic Affairs, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90048
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 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 |
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I. Introduction |
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TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
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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.
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II. LIF—Gene Structure and Regulation |
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TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
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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
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III. LIF Receptor—Gene Structure and Regulation |
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TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
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-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.
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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).
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IV. LIF Signaling |
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TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
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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.
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V. LIF—Hematopoietic and Neuropoietic Cytokine |
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TopAbstractI. IntroductionII. LIF—Gene Structure and...III. LIF Receptor—Gene...IV. LIF SignalingV. LIF—Hematopoietic and...VI. LIF and Endocrine...VII. Integrative...References |
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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.
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VI. LIF and Endocrine Systems |
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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 su
