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Division of Endocrinology (E.A.N.), Department of Medicine, Brown University School of Medicine, Rhode Island Hospital, Providence, Rhode Island 02903; and Yale University School of Medicine and Connecticut Veterans Affairs Healthcare System (K.A.S.), Department of Psychiatry, Research 151, West Haven, Connecticut 06516
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 Top Abstract I. IntroductionII. Biosynthesis of TRH...III. Function of TRHIV. Function of non-TRH...V. Non-TRH pro-TRH-Derived...VI. TRH and Other...VII. TRH DegradationVIII. Concluding RemarksReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Biosynthesis of TRH...III. Function of TRHIV. Function of non-TRH...V. Non-TRH pro-TRH-Derived...VI. TRH and Other...VII. TRH DegradationVIII. Concluding RemarksReferences |
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TRH, produced in the paraventricular nucleus of the hypothalamus (PVN),2 stimulates the biosynthesis and secretion of TSH from the anterior pituitary (8 9 ). TSH, in turn, stimulates thyroid hormone biosynthesis and release (10 ). TRH is central in regulating the hypothalamic-pituitary-thyroid (HPT) axis. TRH influences the release of other hormones, including PRL, GH, vasopressin, and insulin (11 12 13 ), and the classic neurotransmitters noradrenaline and adrenaline (14 ). Further, TRH is present in many brain loci outside of the hypothalamus, supporting a potential role as a neuromodulator or neurotransmitter outside of traditional HPT axis function (15 16 ). For example, TRH is implicated as a modulator of seizure activity (17 ) and gastrointestinal function (18 ). TRH has been also found outside the central nervous system (CNS) in the gastrointestinal tract, pancreas, reproductive tissues including placenta, ovary, testis, seminal vesicles, and prostate, retina, and blood elements (19 ). The widespread distribution of TRH within and outside the CNS supports a diverse range of roles for this molecule, roles likely to involve many functions outside of the traditional HPT axis.
Of the many peptide products derived from the TRH precursor (pro-TRH
described below), until recently only TRH itself was studied
extensively. In the last few years, a new wave of research has
identified many other products derived from pro-TRH and suggests
potential biological functions for these non-TRH peptides. The
immediate precursor to TRH, TRH-Gly, independent of conversion to TRH,
stimulates gastric acid secretion in a dose-dependent manner, although
it is 100-fold less potent than TRH (18 ). Other peptides derived from
pro-TRH, originally referred to as cryptic peptides because their roles
are incompletely understood, are now being studied. In the
hypothalamus, prepro-TRH160–169 (pST10, also
known as Ps4) and prepro-TRH178–199 (pFE22),
peptides that lie between the third and fourth and the fourth and fifth
progenitor sequences for TRH in the TRH precursor, respectively, are
released from perifused rat hypothalamic slices and the median eminence
(20 ). [The reader should note that in Fig. 1
, pro-TRH-derived peptides are named by
"p" for peptide followed by the single letter amino acid
designation for the first and last amino acid of the peptide, along
with the peptide length in subscript. Where these peptides are first
mentioned, they are followed by the longer prepro-TRH name that
describes their amino acid residue positions within the precursor.]
prepro-TRH160–169 potentiates TSH release from anterior
pituitary and stimulates TSHß gene promoter activity (21 ). This
peptide also potentiates TRH-induced gastric acid secretion when
microinjected into the dorsal motor nucleus of the vagus (22 ). Thus,
prepro-TRH160–169 acts in concert with TRH both within and
outside traditional HPT roles. prepro-TRH178–199 is
proposed to be a CRH-inhibiting factor (Refs. 23 24 but see Ref.
25 ).
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Substantial progress has been made in the last few years in
understanding the biosynthesis and processing of prohormone and
neuropeptide precursors. For pro-TRH (Fig. 1
), we have developed a
model of its processing to mature peptides (29 30 31 ). Like many other
secreted peptides, processing of the primary translation product,
prepro-TRH, begins with removal of the signal peptide during its
passage into the lumen of the rough endoplasmic reticulum (RER). From
our current knowledge, processing of pro-TRH takes place within the
regulated secretory pathway (RSP) (for full definition see
Section IIA). Two recently discovered serine proteases,
which are members of the family of prohormone convertases (PCs), PC1
(SPC3) and PC2 (SPC2), related to subtilisin and the yeast-processing
enzyme Kex 2 (32 33 34 ), are the primary PCs involved in
posttranslational processing of pro-TRH (31 35 36 37 ).
Recent contributions to the understanding of pro-TRH biosynthesis and processing provide a useful framework for uncovering the diversity of function displayed by pro-TRH-derived peptides and the way that this diversity is generated. We will review the current knowledge of pro-TRH processing to the most studied biological end product, TRH, and importantly, other pro-TRH-derived peptides. The importance of understanding pro-TRH processing is then underscored by a comprehensive review of the wide range of potential biological roles subsumed by non-TRH products derived from pro-TRH. Although data are limited, where possible we summarize where these peptides are produced and how their levels might be regulated. We then review the HPT axis, with emphasis of its effects on pro-TRH processing. This is followed by a comprehensive review of the function of extrahypophysiotropic TRH. A review of TRH and other pro-TRH-derived peptide receptors then explains mechanisms of pro-TRH peptide signal transduction. Finally, TRH degradation provides an additional way by which peptide levels are tightly controlled, and this area is reviewed. The degradation of other pro-TRH-derived peptides is not yet understood.
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II. Biosynthesis of TRH and Other pro-TRH-Derived Peptides |
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TopAbstractI. IntroductionII. Biosynthesis of TRH...III. Function of TRHIV. Function of non-TRH...V. Non-TRH pro-TRH-Derived...VI. TRH and Other...VII. TRH DegradationVIII. Concluding RemarksReferences |
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Even though the amino acid structures of vasopressin and insulin had been elucidated in the 1950s (41 ), it was not until the early 1960s that the mechanisms of protein biosynthesis began to be understood and the genetic code was fully defined. In 1964, Sachs and Takabatake (42 ) provided the first evidence that the biosynthesis of vasopressin can be inhibited with puromycin, a protein synthesis inhibitor, and that newly synthesized vasopressin could not be detected in tissues until more than 1 h after pulse labeling. In these studies, Sachs and Takabatake demonstrated that before vasopressin becomes a biologically active peptide, it exists in a modified, or proform, state. Posttranslational modification was then required to convert the proform into an active peptide.
While the structure of insulin peptides was described early on (41 ), it
was difficult to envision how the combination of A and B chains was
attained in ß-cells of the pancreas. Experiments initiated by Steiner
in 1965 using tritiated leucine and phenylalanine to label
proinsulin and insulin represented a landmark in prohormone theory.
Using a pancreatic insulinoma derived from a patient, it was possible
to determine that insulin could be derived in vitro from a
single molecule that was converted to the A and B chains by trypsin
treatment (43 ). Studies done in rat islets subsequently demonstrated
conversion of proinsulin to insulin, in a relatively slow process
taking approximately 40 min (44 ). During the same period, work done by
Howell and Taylor (45 ) on insulin biosynthesis showed that newly
synthesized insulin was released several hours after its biosynthesis.
The emerging view of these findings was that some sort of orderly
vectorial transport occurred involving the RER, the Golgi complex (GC),
and secretory granules (SG). These data (46 ), along with the major
contributions of other investigators who established that the
biosynthesis of serum albumin, PTH, and glucagon also originate from
larger precursors, formed the basis of the prohormone theory. This
theory states that synthesis of peptide hormones and neuropeptides
begins with mRNA translation into a large, inactive precursor peptide,
followed by limited posttranslational proteolysis to release bioactive
end products. Chretien and Li (47 ) also made an important contribution
to the prohormone theory when they determined the amino acid sequences
of ß-lipotropin (ß-LPH), 
-LPH, and ß-melanotropin (ß-MSH).
They observed that ß-MSH was part of the ß-LPH sequence, providing
evidence that ß-MSH was a conversion product of ß-LPH. They also
observed that cleavage occurred at the C-terminal side of paired basic
lysine or arginine residues. More definitive evidence for a
precursor/product was provided with the cloning of POMC (29 kDa), which
revealed that the ACTH and ß-LPH sequences were present within the N
terminus of POMC (38 ).
In summary, the biochemical processing of neuropeptides, peptide hormones, and other secreted proteins begins with limited posttranslational proteolysis of larger inactive precursors. Prohormones generally have their hormone sequences flanked by a single, a pair or four (tetra) basic amino acids where subtilisin-like processing enzymes produce their initial endoproteolytic cleavage (48 49 ). This endopreoteolytic cleavage is produced at the C-terminal side of the single or paired basic amino acid residue(s) which is followed by removal of the basic residue(s) by carboxypeptidase enzymes (CP) (50 51 ). Further modifications can occur in the form of N-terminal acetylation, pyroglutamate formation, or C-terminal amidation, which confers bioactivity to many peptides (52 ).
At the intracellular level, hormone precursors are synthesized on membrane-bound ribosomes, by which they are translocated into the lumen of the RER via a signal recognition peptide. During vectorial transport through the GC and beyond, the newly synthesized proteins are subjected to posttranslational modifications including glycosylation, phosphorylation, amidation, acetylation, and proteolytic conversion (48 ). Ultimately, partially processed proteins reach the last compartment of the GC, the trans-Golgi Network (TGN). At the TGN, unprocessed or partially processed products are sorted to the RSP (29 53 54 ) to be stored in immature secretory granules (ISG). Upon maturation, electron-dense SGs containing sorted products can fuse with the plasma membrane in response to an extracellular stimulation in a calcium-dependent manner, thereby releasing their contents into the external milieu (55 ). Two pathways of unstimulated release are proposed for AtT20 cells: constitutive (nongranular) secretion and basal release from compartments that form after sorting into the RSP (56 ). The mechanism whereby constitutive and regulated proteins are differentially sorted into separate vesicles after budding from the TGN is still under intensive investigation (57 58 ).
Two hypotheses have been proposed to explain how proteins are selectively targeted from the TGN to the RSP. In the first hypothesis, proteins are sorted by passive aggregation, in which the proteins condense within forming ISG, thereby excluding other proteins from entering in the granule. This process occurs under acidic pH and high calcium concentrations (59 60 ). Support for the aggregation hypothesis comes from studies done with chromogranin A (61 62 ), chromogranin B (62 ), carboxypeptidase E (CPE) (63 ), and prohormone convertase 2 (PC2) (64 ). However, there are data suggesting that aggregation alone is not sufficient for sorting. Modifications of the chromogranin B sequence (65 ) can prevent the correct sorting of these peptides to the RSP, while their in vitro aggregation properties appeared unaltered. The insulin-like growth factor-1 (66 ) does not aggregate in the TGN, but is still sorted in the RSP.
The second hypothesis, originally proposed in 1985 by Kelly (67 ),
involves cis-acting sorting signals within a protein
destined for the RSP that interact with membrane-bound sorting
receptors. Sorting receptors, possibly located in the forming SG,
direct segregation of the protein for further packaging into SGs.
Protein aggregation within the ISG can occur in this model, but is more
critical for product concentration than sorting per se.
Evidence supporting this second hypothesis has come from experiments
involving chimeric proteins (68 69 70 71 72 73 ), where the fusion of
constitutively secreted protein to a protein destined for RSP caused a
rerouting of this protein to the RSP. Conversely, proteins that have
their sorting signal domains modified may be misrouted from the RSP
into the constitutive pathway, as demonstrated for POMC (74 ),
chromogranin A and B (75 ), PC2 (69 ), and glycine 
-amidating
monooxygenase (PAM) (76 ). A related hypothesis is sorting by retention,
in which all proteins are initially targeted to ISGs, after which
proteins that do not belong in the RSP are removed to their final
destination, e.g., lysosomal enzymes (57 77 ).
It has been proposed recently that the membrane form of CPE, localized to the TGN, is a sorting signal receptor (78 ). CPE is proposed to direct POMC, proinsulin, proenkephalin, but not chromogranin A, into the RSP (79 ). Thus, CPE is a common sorting receptor for some, but not all, prohormones, and there must be other sorting receptors to direct trafficking of other proteins to the RSP (79 ). However, Irminger et al. (80 ) have provided evidence refuting the claim that CPE is a sorting receptor for proinsulin. In those studies they used pancreatic islets isolated from CPE-deficient (Cpefat/Cpefat) and control (Cpefat/+) mice to examine whether the trafficking of proinsulin and insulin was affected. They found that CPE was not essential for the sorting of proinsulin to the RSP (80 ). However, similar experiments with procholecystokinin in Cpefat/Cpefat mice indicate that CPE does function as a sorting receptor (81 ).
With this as a background, we turn specifically to the processing of
pro-TRH. The elucidation of the rat prepro-TRH sequence in 1986 was of
key importance to understanding the processing of pro-TRH to TRH and
non-TRH peptides. Rat prepro-TRH is a 29-kDa polypeptide composed of
255 amino acids. The rat precursor contains an N-terminal 25-amino acid
leader sequence, 5 copies of the TRH progenitor sequence
Gln-His-Pro-Gly flanked by paired basic amino acids (Lys-Arg or
Arg-Arg), 4 non-TRH peptides lying between the TRH progenitors, an
N-terminal flanking peptide, and a C-terminal flanking peptide (82 83 ). The N-terminal flanking peptide
(prepro-TRH25–50-R-R-prepro-TRH53–74) is
further cleaved at the C-terminal side of the arginine pair site to
render prepro-TRH25–50 and prepro-TRH53–74,
thus yielding a total of 7 pro-TRH-derived peptides (Fig. 1).
The biosynthesis of TRH and other pro-TRH-derived peptides follows the same prohormone-processing mechanisms described above, beginning with mRNA-directed ribosomal translation, followed by posttranslational limited proteolysis of the larger precursor, proTRH. This process occurs while pro-TRH is transported from the TGN to newly formed ISGs (84 ). These granules then mature and are targeted to sites of secretion at the plasma membrane of the cell. Rat, mouse, and human pro-TRH, similar to other peptide hormone precursors such as pro-enkephalin, contains multiple copies of one of its peptide products, in this case, the progenitor for TRH, Gln-His-Pro-Gly. Most of the products derived from pro-TRH are targeted into the RSP. Cleavage of the precursor to generate biologically active TRH occurs at paired basic residues by the action of PC1 and PC2 (31 35 37 ) followed by the action of CPE to remove the basic residue(s) (85 ). Gln-His-Pro-Gly is then amidated by the action of PAM, which uses the C-terminal Gly as the amide donor, and the Gln residue undergoes cyclization to a pGlu residue to yield TRH.
In the last few years, this laboratory has elucidated the processing steps involved in the synthesis of TRH and pro-TRH-derived peptides. An understanding of the biosynthesis and processing of pro-TRH is critical to appreciating how, when, and where modulation of this central regulator of the HPT axis takes place during physiologically appropriate modulation of thyroid function. It is also critical to understanding the function of TRH and other pro-TRH-derived peptides in extrahypothalamic regions of the brain, or outside of the nervous system, as discussed in Section VI of this review. Initial studies of TRH biosynthesis were difficult because of the low levels of TRH and other pro-TRH-derived peptides produced in hypothalamic tissue in vivo. In the search for a better system to perform these studies, initial work was done by stably transfecting prepro-TRH cDNA into transformed cell lines (86 87 ) to attain higher levels of pro-TRH expression. Among the cell lines investigated, only AtT20 (corticotroph, mouse) and RIN 5F (insulinoma, rat) cells were able to efficiently cleave the TRH precursor at paired basic amino acid residues to generate mature TRH, as well as pro-TRH-derived peptides, which were identical to those previously identified in vivo (88 89 ). GH4C1 (somatommamotroph, rat) and 3T3 (fibroblast, rat) cells were unable to process the pro-TRH precursor. When these studies were performed, little information was available about PCs, their role in prohormone processing, or their expression in different tissues and cell lines. Retrospectively, it was determined that the cell lines capable of processing proTRH, AtT20 and RIN 5F cells, also express PC1 and PC2 (90 ).
A model of pro-TRH posttranslational processing was developed in
experiments with transfected AtT20 cells expressing rat
prepro-TRH (30 ). By means of Western blot analysis, immunoprecipitation
followed by SDS-PAGE, and RIA, it was determined that pro-TRH is
present in transfected AtT20 cells and in primary cultures
of hypothalamic neurons, an endogenous source of pro-TRH, as a 26-kDa
protein (37 ). Pulse-chase studies indicated that the 26-kDa precursor
is cleaved at two mutually exclusive sites to generate the first
intermediate forms (Fig. 1). One cleavage generates a 15-kDa N-terminal
peptide (prepro-TRH25–151 or 157) and a 10-kDa C-terminal
peptide (prepro-TRH154 or 160–255). An alternate cleavage
generates a 9.5-kDa N-terminal peptide (prepro-TRH25–106 or
112) and a 16.5-kDa C-terminal peptide (prepro-TRH109 or
115–255). These cleavage steps occur in the TGN (Fig. 2),
before packaging into ISGs (29 84 ), in agreement with similar studies
of the cellular location of early processing for POMC and somatostatin
(SRIF) (53 ).
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). Processing of the 9.5-kDa N-terminal fragment
arising from the alternative cleavage of the 26-kDa prohormone at
residues 107–108 is postulated to result in the production of the
N-terminal peptides, prepro-TRH25–50,
prepro-TRH53–75, and prepro-TRH83–106, while
the 16.5-kDa fragment is processed to produce the 5.6-kDa
prepro-TRH160–199 and the 5.4-kDa
prepro-TRH208–255 (Fig. 1).
Recent experiments confirm the proposed model that the C-terminal
10-kDa peptide derived from an initial cleavage at residues
Lys152-Arg153 is the precursor to two peptides
of 5.6- (prepro-TRH160–199) and 5.4-kDa
(prepro-TRH208–255) (Fig. 1). After the cleavage of basic
residues at positions 199–200 or 207–208, the 5.6-kDa peptide is
further processed to generate the prepro-TRH160–169 and
prepro-TRH178–184. prepro-TRH178–184 is
further cleaved to two novel peptides, prepro-TRH178–184
and prepro-TRH186–199 (91 ), Fig. 1).
B. Intracellular sites of pro-TRH processing
Initial studies in 1993 showed that processing of the 26-kDa
pro-TRH precursor to smaller intermediates occurred before packaging
into ISGs (29 ). The experimental strategy used to more precisely define
the intracellular sites of pro-TRH processing was to block peptide
transport from one cellular compartment to another and to characterize
processing that occurred before the point of blockade (84 ). To study
processing that occurs in the RER, peptides were blocked in their exit
from this compartment with brefeldin A (BFA) treatment of
AtT20 cells expressing the prepro-TRH cDNA (30 87 ). BFA is
a fungal metabolite that blocks ER-to-Golgi transport of proteins by
reversibly inhibiting the exchange of GDP for GTP in the GTP-binding
protein that is a key component in the vesicular transport, the ARF
protein. This prevents protein recruitment to intracellular membranes
and inhibits subsequent vesicle formation. To study the processing
steps that occur in the GC, cells were incubated at reduced
temperatures. Incubation of cells at 20 C prevents packaging of
proteins into ISGs at the TGN and has been used previously to study the
processing of other prohormones (53 92 ). Figure 2 summarizes the results of these
blockade experiments, described more fully below.
When transfected AtT20 cells expressing prepro-TRH were treated with BFA to block ER-to-GC transport, the 26-kDa pro-TRH precursor accumulated 4-fold over control levels, indicating a significant degree of post-ER processing. However, some processing of 26-kDa pro-TRH to the 15-kDa and the 9.5-kDa N-terminal intermediates, and the 16.5-kDa and 10-kDa C-terminal intermediates, was seen. The accumulation of the 16.5-kDa intermediate was not as great as that of the 15-kDa intermediate when compared with control levels (4-fold for the 15 kDa and about 2-fold for the 16.5 kDa) (84 ). To further clarify where the initial site of pro-TRH cleavage occurred, a combination of temperature blockade and BFA treatment was performed. With this strategy it was demonstrated that the 26-kDa precursor protein (accumulated 4-fold) is processed in the GC (possibly in the TGN) to generate the 15-kDa/10-kDa and the 9.5-kDa/16.5-kDa intermediate pairs. When the fate of the 15- and 16-kDa intermediates was analyzed, while they were retained within the TGN at 20 C, the 16-kDa C-terminal intermediate was further processed at basic residues 206–207 to the 5.4-kDa C-terminal peptide. In contrast, the 15-kDa N-terminal intermediate appeared to undergo processing in a post-GC compartment, i.e., the SGs. This observation strongly suggests that these two intermediates follow different paths of processing (84 ).
Evidence supporting differential distribution for the N- and C-terminal
peptides comes from recent immunocytochemical (ICC) studies using
transfected AtT20 cells, which indicate that pro-TRH, as
well as the 15- and the 6-kDa N-terminal intermediates, are located in
the GC and TGN (Fig. 3A). In
contrast, end products, including
prepro-TRH25–50, prepro-TRH160–169,
and TRH, are only present in SGs along the plasma membrane and in cell
processes (Fig. 3F). C-terminally directed antisera that recognize
pro-TRH and the 16.5- and 5.4-kDa C-terminal peptides result in
positive immunostaining in the GC, along the plasma membrane, and in
cell processes (Fig. 3H). Thus, C-terminal intermediates appear to
reach further along the RSP before processing than their N-terminal
counterparts. This differential processing might serve as a mechanism
to regulate the timing of production of peptides such as
prepro-TRH160–169, prepro-TRH178–199, and
prepro-TRH53–75, and possibly TRH. For example, the
16.5-kDa intermediate, which is processed in the TGN, contains
prepro-TRH178–199 and preproTRH160–169. A
portion of such peptides, formed before their entry into SGs, might
exit the cell via the constitutive pathway to maintain a basal level of
release independent of TRH secretion.
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, B and
E). Immunoelectron microscopy (IEM) confirms positive staining in the
GC (Fig. 3E). Some ISGs budding from the TGN-like were also
stained. Since positive staining was detected in boutons, it was
proposed that these intermediates are processed to mature
pro-TRH-derived peptides near the axon terminal, before secretion. ICC
using anti-TRH, anti-prepro-TRH53–75, and
anti-prepro-TRH160–169 showed positive staining only in
the neuronal processes and axon terminals while the soma remained
unstained (Fig. 3G). The positive staining observed in neuronal
processes and axon terminals with these antibodies was confirmed by IEM
(Fig. 3G, inset).
Note that in transfected AtT20 cells using the same
antiserum, immunostaining was seen only in GC and ISGs, but not in
cellular processes (Fig. 3A). ICC on transfected AtT20
cells using anti-TGN38, a TGN marker, indicated that the
TGN can be projected away from the GC toward budding ISGs (Fig. 3C).
Thus, in AtT20 cells processing of the 15-kDa N-terminal
peptide takes place somewhere between the TGN and ISGs, suggesting that
prepro-TRH25–50, prepro-TRH53–75, and
prepro-TRH83–106 are already formed by the time SGs
mature.
Using the C-terminal antiserum that recognizes pro-TRH and the 16.5-,
10-, and 5.4-kDa peptides, positive staining was visualized in a patchy
cytoplasmic distribution (Fig. 3I), often closely associated with the
nucleus. Immunoreactivity was also observed in neuronal boutons, axon
terminals, and unbranched growth cones. IEM confirmed that the patchy
cytoplasmic areas were within the ER and GC (36 ) (Fig. 3I, inset); all layers of the GC (cis, medial, and
trans) were immunostained with this antibody (36 ). The
contrasting staining patterns for the two antisera (N- and C-terminal)
(Fig. 3, B and I) suggest the existence of a different peptide
distribution for N-terminal vs. C-terminal peptides,
possibly due to different intracellular routing of intermediates to
SGs. However, no conclusive data are yet available to confirm this
hypothesis.
C. Tissue-specific processing of pro-TRH
Increased knowledge of differential processing has led to a better
understanding of how multiple biological peptides, with different
functions, are generated from the same prohormone. This concept is
further reinforced by the observation that certain regions in the brain
can give rise to several different pro-TRH-derived peptides in addition
to, or instead of, TRH. This will become clear in a review of the
neuroanatomical distribution of pro-TRH and pro-TRH-derived peptides.
TRH-positive axons in the ME originate from neuronal perikarya in the PVN. The PVN is composed of two major components, the magnocellular and the parvocellular divisions. The parvocellular division contains most of the TRH neurons that project to the ME. A large population of immunoreactive neurons is located in medial and periventricular parvocellular subdivisions, organized in a triangular configuration, symmetric to the dorsal aspect of the third ventricle, whereas the anterior parvocellular subdivision neurons are more disperse (93 ). However, not all TRH-containing neurons in the PVN project to the ME (94 ). In addition to the PVN, TRH neurons are present in many other regions of the hypothalamus (93 ). As these populations of neurons have no known projections to the ME, and are not regulated in conjunction with the thyrotropic neurons of the PVN, it is presumed that they do not subserve a direct hypophysiotropic function.
The largest concentration of hypothalamic TRH neurons outside of the
PVN are found in the dorsomedial nucleus, lateral hypothalamus, and
preoptic area, including medial, periventricular, suprachiasmatic, and
the sexual dimorphic nucleus of the preoptic area (93 ). In addition to
the PVN, TRH neurons are present in many other regions of the CNS
including regions in the diencephalon, telencephalon, mesencephalon,
myelencephalon, and spinal cord (Table 1). An extensive anatomic description of
TRH neurons and TRH fibers in these tissues has been reported
previously (93 ). While the presence of TRH in these regions is clear,
relative levels are largely unknown because most mapping has depended
on ICC detection in animals pretreated with colchicine. Detailed
microdissection studies, combined with RIA, have been done for some
areas, including the brainstem and hippocampus. These studies do not
require colchicine pretreatment, are more quantitative than ICC
detection, and reveal an even broader distribution for TRH than
appreciated by ICC. However, their neuroanatomical resolution falls
short of that of ICC, and these latter studies are the primary source
of TRH data presented in Table 1.
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). In the case
of pro-TRH, the reticular nucleus of the thalamus contains abundant
prepro-TRH mRNA and several pro-TRH-derived peptides in their extended
forms, but does not contain mature TRH (95 ). Moreover, the N-terminal
extended forms of TRH, TRH-prepro-TRH160–169 and
TRH-prepro-TRH178–199 are major end products of pro-TRH
processing in the olfactory lobe (OB) (96 97 ), but not in the
hypothalamus where pro-TRH is completely processed to non-TRH peptides
and TRH (96 ). In the ME, PVN, and preoptic area (POA), pro-TRH is fully
processed to its mature forms, while in the OB less than 60% of
N-terminal prepro-TRH25–50 is formed. Similarly, while in
the OB the 10-kDa C-terminal intermediate (see Fig. 1) is the main end
product, in the ME and PVN this intermediate is fully processed to
prepro-TRH160–169 and prepro-TRH178–199 (Fig. 1). Finally, in the POA partial processing of the 10-kDa peptide is
observed, and the lateral hypothalamus contains lower levels of both N-
and C-terminal pro-TRH-derived peptides as compared with the ME and POA
(E. A. Nillni, unpublished data). Table 1 summarizes the current
information regarding neuroanatomical distribution of intermediate
forms of pro-TRH processing, mature pro-TRH-derived peptides, and TRH
peptides in perikarya and fibers.
D. The role of PCs and CPs
The PCs are a family of seven subtilisin/kexin-like endoproteases
including furin, PC1 (also known as PC3), PC2, PC4, PACE4, PC5-A (also
known as PC6-A), its isoform PC5-B (also known as PC6-B), PC7 (also
known as LPC), and PC8 (also known as SPC7) (32 33 34 98 99 100 101 )). The
structure of these serine proteinases resembles both the bacterial
subtilisins and yeast kexin (90 102 103 ). These enzymes cleave at the
C-terminal side of single, paired, or tetra basic amino acid residue
motifs (104 ), followed by removal of remaining basic residue(s) by CPs
(50 51 ). The selective expression of PC1 and PC2 in endocrine and
neuroendocrine cells suggests they are significant in prohormone
processing (32 90 98 105 ). PC1 and PC2 have been shown to process
pro-TRH (31 35 37 106 ), proinsulin (104 107 108 ), proenkephalin
(109 ), prosomatostatin (110 111 ), and POMC (112 113 ) to various
intermediates and end products in coexpression experiments.
Like their substrates, PC1 and PC2 undergo maturation from larger precursor proteins. Maturation of pro-PC1 begins in the ER and continues in the TGN (90 ). In contrast, pro-PC2 maturation begins in the TGN and continues in the SGs; active PC2 and PC1 accumulate in SGs (114 115 ). PC2 has been proposed to produce smaller peptides from intermediates resulting from cleavage by PC1 (109 112 113 ). Consistent with this, PC1 is implicated in the early cleavage of POMC (112 113 116 ) and proinsulin (117 ). Thus, the compartments where these enzymes are active also are determined by differential timing of their respective maturation.
The distribution of neurons containing mRNAs encoding prepro-TRH, PC1,
and PC2 in the PVN and other areas of the brain has been determined
using in situ hybridization (106 ) (Table 1). The glomerular
layer of the OB displays coexpression of prepro-TRH with PC2 mRNA, but
not PC1, whereas in the tenia tecta coexpression of mRNA for prepro-TRH
with PC1, but not PC2, is evident. The PVN displays prepro-TRH mRNA
coexpression with both enzymes, whereas the lateral hypothalamus shows
coexpression of prepro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Double
in situ hybridization indicates that in the PVN, PC2 mRNA is
present in 60–70% of TRH neurons, and PC1 is present in 37–46% of
TRH neurons (118 ). Even though these investigators found a trend for
more coexpression of mRNA for prepro-TRH with PC2 than PC1,
coexpression alone does not define which enzyme is more important in
the processing of pro-TRH in vivo.
During the last few years, Nillni’s laboratory has provided unequivocal evidence for the role of PC1 and PC2 in the processing of pro-TRH (31 35 36 37 ). In coinfection experiments, where recombinant vaccinia viruses are used to coexpress PC1, PC2, PACE4, PC5-B, and furin, together with rat prepro-TRH in constitutively-secreting LoVo cells or in the regulated-secreting endocrine GH4C1 cell line, RIA of LoVo-derived secreted products demonstrates that furin cleaves the precursor to generate both N- and C-terminal intermediates, while PC5-B does not produce any peptide. PC1, PC2, and PACE4 only produce N-terminal intermediates, and less efficiently than furin. Interestingly, in LoVo cells, furin cotransfection produces TRH-Gly at much greater levels than any of the other PC enzymes. Recent data indicate that furin, which is ubiquitously expressed in all tissues, may serve a role in processing of prosomatostatin within the constitutive pathway (110 111 ). Since LoVo cells only contain the constitutive secretory pathway, these results suggest that pro-TRH can be processed to a certain extent without entry into the RSP. However, caution must be taken with this interpretation because under conditions of viral expression, the unusually high level of virus in coinfected cells can produce disruption of cellular compartments. The products resulting from coexpression of prepro-TRH with either furin or PC1 are similar, in agreement with their similar specificity observed in a number of cell coexpression experiments (109 111 ) and in vitro data (119 ).
In GH4C1 cells, PC1, PC2, furin, PC5-B, and PACE4 produce
both N-terminal and C-terminal peptides derived from pro-TRH.
Significantly, TRH-Gly and TRH are produced in highest amounts by PC1,
PC2, and furin. Further analysis of the cleavage specificity of PC1 and
PC2 reveals that PC1 is primarily responsible for cleavage of the
entire TRH precursor to mature TRH, as it can generate all products at
significantly higher levels than PC2 (Fig. 4). While 7B2 is known to be involved in
the maturation of PC2 (Fig. 4) (90 120 ) it does not augment the
ability of PC2 to cleave pro-TRH to either N- or C- terminal forms.
Subsequently, we have examined the role of PC1 and PC2 in the formation
of prepro-TRH178–199 by coexpressing rat prepro-TRH cDNA
with PC1, PC2, and 7B2 in GH4C1 cells (91 ). PC1
effectively cleaved pro-TRH to immunoreactive forms recognized by
anti-prepro-TRH178–199, while PC2 played a minor role,
even in the presence of 7B2 (Fig. 1).
|
) (37 ).
|
-Ser186-Trp187-Glu188-Glu188-Lys189...
) generating two novel peptides, pFQ7 and pSE14
(Fig. 1).
These two novel peptides, prepro-TRH178–184 and
prepro-TRH186–199 are present in rat PVN, lateral
hypothalamus, and ME (91 ). Thus, the antibody generated against the
prepro-TRH178–199 sequence recognizes the 10-kDa peptide,
a 5.6-kDa form that probably is prepro-TRH160–199 (30 ), a
2.6-kDa peptide that is prepro-TRH178–199, and two smaller
moieties of 1.6 and 0.84 kDa that are proposed to be
prepro-TRH186–199 and prepro-TRH178–184,
respectively (Fig. 1). Figure 6 shows a
diagrammatic representation of rat pro-TRH and its cleavage by PC1 and
PC2 as proposed from the most recent studies and compared with previous
in vitro studies (31 35 91 ).
|
combines current neuroanatomical distribution data for
prepro-TRH mRNA, pro-TRH, and pro-TRH-derived products with the
distribution of PC mRNAs and enzymes. Also included is a summary of
pro-TRH processing in transfected cells and primary cultures of
hypothalamic neurons and pituitary cells. Several important conclusions
can be drawn. pro-TRH is widely distributed in the hypophysiotropic and
extrahypophysiotropic areas of the brain. The widespread expression of
pro-TRH, PC1, and PC2 mRNAs, with their overlapping distribution in
many areas of the rat CNS, indicates the striking versatility provided
by tissue-specific processing in generating quantitative and
qualitative differences in non-TRH peptide products as well as TRH.
Examples of these differences for several tissues are presented in the
first column of the table and described above. A most striking example
is the reticular nucleus of the thalamus, where PC1 and PC2 are not
coexpressed with pro-TRH. TRH is not produced in this nucleus,
indicating a central role for PC1 and PC2 in maturation to TRH.
However, other pro-TRH intermediates are present in the reticular
nucleus, suggesting PCs other than PC1 and PC2 might be involved in
processing of pro-TRH for this particular region of the CNS (106 ).
Differential processing has been reported for other prohormones, and
these differences relate to alterations in the expression of various
PCs within different cell types. POMC is processed primarily to ACTH,
ß-endorphin, and N-POMC1–77 in the anterior pituitary
(melanotrophs). In turn, these products are further processed to
-MSH, ß-endorphin1–31, N-POMC1–49,
and -MSH in the intermediate lobe and brain (38 ). Differential
processing of a common polypeptide precursor is dependent upon the
processing enzymes expressed in each specific cell type. Proenkephalin,
which contains seven identical copies of met-enkephalin, is processed
to large intermediate forms in the adrenal medulla, whereas this
precursor is cleaved primarily to the pentapeptide met-enkephalin in
the brain (39 ). The biological actions of substance P (SP) depend on
the enzymatic processing of its precursor by the processing enzymes
prolylendopepetidase to yield SP5–11, and endopepetidase
3.4.24.11 to yield SP1–7. While SP1–7 acts as
an analgesic, inhibits aggression, and enhances learning and memory,
the SP5–11 enhances pain transmission, stimulates
aggression, and blocks learning and memory (40 ). In the brain,
procholecystokinin (pro-CCK) is processed to produce only CCK8 amide,
while in the gut the precursor is cleaved to larger molecules, such as
CCK12, 22, 33, 38, 58, and 83 amide (121 ). Transfection experiments
have shown that proneuropeptide Y (pro-NPY) can be cleaved by cell
lines expressing either PC1 or PC2, but pro-NPY is primarily processed
by PC2 in superior cervical ganglia (122 ). Thus, differential
processing of neuropeptitdes including pro-TRH, pro-NPY, POMC, pro-CCK,
SP, and proenkephalin provides a critical mechanism through which cells
regulate the levels of specific peptides to fulfill different
physiological requirements, a mechanism potentially more versatile than
the alternative splicing of mRNA.
As mentioned in Section IIA, CPs remove remaining C-terminal basic residues from prohormone intermediates that are initially cleaved by PCs. Experiments with the fat/fat mouse model of CPE deficiency (123 ) support a role for CPE in the processing of pro-TRH (85 ). Mice homozygous for the fat/fat mutation are obese, diabetic, and infertile. These mice have a missense (Ser to Pro) mutation at CPE residue 202 that abolishes enzymatic activity (123 ). Hypothalamic TRH levels are depressed 65% in fat/fat mice relative to heterozygous controls. SDS-PAGE demonstrates hypothalami from both wt/fat and fat/fat mice contain moieties different from those of the wt/wt mice. Specifically, 6.5-, 3.0-, 2.6-, and 1.6-kDa forms of the pro-TRH sequence are detected, and their levels differ significantly between the two groups. Compared with wt/fat mice, fat/fat mice hypothalami contain 20-, 3-, and 2-fold elevations in the 6.5-, 3.0-, and 2.6-kDa species. These data indicate that the fat/fat mutation produces qualitative changes in pro-TRH processing, and that CPE is involved in the later stages of pro-TRH processing. However, cell transfection experiments would help to rule out secondary phenotypic changes caused by the CPE mutation in these mice. Further, since hypothalami from fat/fat mice contain immunoreactive TRH, additional CPs must also be able to process pro-TRH to TRH, assuming that the TRH detected is not a cross-reactive non-TRH species. CPs such as carboxypeptidase D, with similar enzymatic properties to CPE, are also present in compartments of the secretory pathway and are distributed in many tissues, including the brain (50 ).
Interestingly, in fat/fat mice, levels of TSH,
T3, and T4 were normal, suggesting that 34% of
normal TRH levels is sufficient to maintain the thyroid function. This
last observation is important because it is hypothesized that the five
identical progenitor sequences of TRH contained in the prohormone may
not be processed to mature TRH at all times, and that only a few
of them may be needed to maintain the thyroid function. In cultured
hypothalamic cells at steady state, previous studies had shown that the
ratio of mature TRH to prepro-TRH25–50 and the 5.4-kDa
C-terminal peptide (Fig. 1) is 3:1 instead of the theoretical 5:1 for
complete processing (36 ). A similar ratio is seen in transfected
AtT20 cells (30 ). In the rat brain, the ratio of TRH to
other pro-TRH-derived peptides sequences is almost 1:1 in the
hypothalamus, and 1.5:1 in the olfactory lobe (89 ). The above ratios
are indicative of incomplete yields of TRH from pro-TRH, although only
if degradation rates do not contribute significantly to these various
ratios. Further, the relative immunoreactivites of various antisera
used to their iodinated tracers has not been defined, leaving exact
molar ratios difficult to calculate. Still the range of TRH to
pro-TRH-derived peptides found in various tissues makes it likely that
full TRH yields are not achieved in all, or even most, tissues.
If not all TRH progenitor sequences are cleaved from pro-TRH, the resulting TRH progenitor sequences, linked to amino- or carboxyl-terminal extensions, would not be detected in usual TRH RIAs. The TRH assay is specific for mature TRH, needing both the amino-terminal pyro-Glu and carboxy-terminal Pro-amide for detection. Nonimmunoreactive TRH progenitors might also retain untrimmed basic amino acid residues, carboxyl-terminal glycine residues, etc. Another explanation for the lower than 5:1 ratio of TRH to other products could be that TRH is extracted less efficiently from cells than these other peptides, although this seems unlikely because TRH is the smallest of the peptides, and doping experiments indicate TRH is extracted with high efficiency under our conditions. As described above, it is proposed that the processing of peptides derived from the N-terminal portion of pro-TRH is substantially different from the processing of those of the C-terminal end. This suggests the possibility of differential maturation of TRH molecules depending on location within the pro-TRH sequence (84 ). If this is the case, either the three TRH molecules derived from the C-terminal side, or the two from the N-terminal side, may play a more primary role in hormonogenesis. It is also possible that certain TRH molecules become biologically active at different times than others, or only some of them reach maturity while the rest are degraded by the TRH-degrading enzymes, depending upon physiological needs (see Section VII). Finally, cells may simply produce excess TRH that may or may not be used.
In summary, we have presented an overview of the current knowledge of pro-TRH biosynthesis, its processing, its tissue distribution, and the role of known processing enzymes in pro-TRH maturation. Evidence is presented suggesting differential processing for pro-TRH at the intracellular level is physiologically relevant. The data indicate that PC1 is primarily responsible for most pro-TRH cleavage events. PC2 is involved in specific processing events that occur later in the secretory pathway, specifically in the formation of the second TRH molecule from the N-terminal side of prepro-TRH83–106, and the proteolytic cleavage of prepro-TRH178–199 to generate the novel prepro-TRH178–184 and prepro-TRH186–199 peptides.
E. Neuropeptide and catecholamine regulation of pro-TRH
biosynthesis and processing
Immunoreactive TRH (iTRH) axon terminals are present in high
density in the external layer of the rat ME, in close apposition to
capillaries of the hypophysial-portal system (124 ). These axons
originate from neuronal perikarya located in the PVN, the
"thyrotrophic area" of the rat hypothalamus. Destruction of this
region results in disappearance of up to 94% of TRH in the external
layer of the ME and reduction of TSH secretion from the anterior
pituitary gland (125 ). As described in Section IIC (Table 1), in addition to the PVN, iTRH neurons are present in other regions
of the hypothalamus, including the POA, anterior hypothalamus, and
supraoptic, arcuate, dorsomedial, and premmamilary nuclei, as well as
basolateral and prefornical hypothalmus (93 ).
Although many neurons in the PVN contain more than one peptide, TRH neurons are unique in being almost always unassociated with other known peptides (126 ). This makes the regulation of pro-TRH-derived peptide biosynthesis very specific. As described in more detail below, TRH neurons in the PVN are located in a region where they can be regulated by a number of neuroendocrine inputs. TRH neurons are densely innervated by norepinephrine (NE)-containing axons that stimulate TRH secretion (124 ). TRH neurons are also densely innervated by neuropeptide Y (NPY) neurons. In smaller numbers, SRIF and endogenous opioid peptide (EOP) terminals are also in contact with TRH neurons (124 ). In vivo, these various neuroendocrine inputs may affect the levels of prepro-TRH mRNA and the posttranslational processing of pro-TRH by influencing the biosynthesis and maturation of PC1 and PC2. At the present time, the effect of this input on pro-TRH processing is unknown. Evidence for coordinated regulation of mRNAs for processing enzymes and their substrates has been documented in several cases (127 ). In contrast, outside the hypothalamus TRH is colocalized with other substances. For example, in the descending bulbospinal pathway, TRH is colocalized with SP and serotonin (5-HT) (128 ). Regulation of these TRH neuronal systems is much less well characterized than the thyrotropic neurons of the hypohyseal-portal system.
TRH-synthesizing neurons in the rat PVN receive a large number of
afferent neuroendocrine inputs. Axon collaterals of parvocellular
neurons ramify within the medial parvocellular PVN and establish
numerous synaptic contacts with perikarya and dendrites of other
parvocellular as well as magnocellular neurons, e.g., SRIF
afferents are derived from the PVN itself (129 ). The majority of inputs
to TRH neurons are derived from the diencephalon, telencephalon, and
brainstem (130 ). The paraventricular and medial parvocellular divisions
of the PVN are densely innervated by NE-containing and epinephrine
(E)-containing inputs from the medulla and pons (131 ). Further,
NE-containing neurons densely innervate the midregion of the external
layer of the ME. These inputs activate tuberoinfundibular neurons.
Intracerebroventricular (icv) injections of NE, E, and 2-adrenergic
agonists stimulate basal TSH secretion (132 133 ), and NE/E treatment
of hypothalamic preparations stimulates TRH release (134 ). Inhibitors
of catecholamine (CA) biosynthesis or 2-adrenergic antagonists lead
to a fall in basal TSH secretion. Thus, NE and E exert a tonic,
stimulatory regulation on TSH secretion principally through
2-adrenergic receptors. Stimulated release from the ME appears to be
postsynaptically mediated via 1-adrenergic receptors (135 ). In
contrast, locus coeruleus (LC) afferents are inhibitory, being
activated during stress (136 ). NE/E excitation of PVN TRH neurons
mediates the rise in TSH in response to acute cold exposure or
hypovolemia (135 137 138 ). However, it has also been proposed that
1-adrenergic receptors mediate a phasic inhibitory regulation of TSH
release. The data on NE/E modulation of TRH biosynthesis may be
reconciled by an examination of how these inputs affect the
posttranslational processing of pro-TRH, as well as examining their
effects on PC biosynthesis. Peripheral levels of T3,
T4, or TSH may also influence NE/E effects on TRH
biosynthesis and/or release.
The PVN also receives prominent dopamine (DA) inputs from the posterior
and dorsal areas of the hypothalamus, the zona incerta of the
subthalamic region, and the A14 region of the anterior hypothalamus
(124 ). Mesencephalic A9 and A10 dopaminergic neurons also project to
the PVN. In turn, large terminal fields to the ME originate in the
arcuate nucleus and periarcuate nucleus regions of the hypothalamus. In
contrast to the NE/E system, DA inputs appear to inhibit TRH secretion,
mainly at the level of the ME (139 ). Augmentation of DA
neurotransmission inhibits basal and/or cold-stimulated TSH
release, while DA antagonism has the opposite effect, although some
studies have failed to replicate these findings (124 ). In addition, TRH
release may be indirectly inhibited by DA-stimulated secretion of SRIF
(140 ). Conversely, DA stimulates TRH release from isolated hypothalamic
fragments (134 140 ), again reinforcing the need to examine TRH
biosynthesis in both in vitro and in vivo
systems. Within the HPT axis, thyroid hormones appear to modulate DA
levels in the ME, and TSH increases the ability of DA to inhibit TRH
(141 ) (Table 1).
A wide array of neuropeptides, including NPY, TRH itself, SRIF, EOPs,
neurotensin (NT), and vasoactive intestinal polypeptide (VIP), have
inputs to the PVN and/or external layer of the ME (142 143 144 145 146 ). Other
mediators, including -aminobutyric acid (GABA) and various
cytokines, also appear to regulate TRH or TSH secretion, but there is
as yet no anatomical evidence to support a direct action on TRH neurons
in the PVN and/or ME (124 ). Anatomically, NPY appears most prominent in
its inputs to the periventricular and medial parvocellular divisions of
the PVN (147 ). NPY cell bodies principally reside in the medulla, often
coexisting with NE and E (148 ), but other sources come from throughout
the brain, including the arcuate nucleus of the hypothalamus itself.
Indeed, the arcuate nucleus is the major source for NPY fibers
innervating the TRH neurons in the PVN (149 ). Few NPY-containing axons
project to the ME. The effects of NPY on tuberinfundibular TRH are not
yet well understood. NPY neurons also innervate SRIF neurons in the
PVN, which would allow indirect regulation of TRH biosynthesis or
secretion (124 ). Central administration of NPY reduces NE utilization
in the PVN, as well as TSH release, indicating an inhibitory
influence. In vivo, NPY also increases hypothalamic DA
content, as well as DA turnover in the ME, the net result of which
would reduce TRH release as well (150 ). Physiologically, NPY is
critical to integrating thyroid function, food intake, and
thermoregulation (151 ) (Table 1).
Inputs containing EOPs represent a second rich innervation to the PVN. These originate in the arcuate nucleus, periarcuate area, and amygdala (144 ). The dorsal raphe projects 5-HT/enkephalin (ENK) axons, and the posterior hypothalamus-mammallary bodies send GABA/histamine/ENK projections, to the PVN. The ME contains numerous ENK, dynorphin, and endorphin (END) synapses originating from the PVN and arcuate nucleus. Both END and ENK inhibit TRH release from the hypothalamus, and ENK and morphine inhibit TRH secretion from the ME (152 ). There is additional evidence that ENK indirectly inhibits tuberoinfundibular TRH via DA release (153 ).
Recent data indicate that pro-TRH processing is regulated by opiate withdrawal (27 ). Opiate withdrawal increases prepro-TRH mRNA, and the N-terminal prepro-TRH53–74 and prepro-TRH83–106 peptides, in the rat PAG, whereas the level of TRH is unaltered (27 154 ). New data also show suckling increases the production of prepro-TRH178–199 and prepro-TRH186–199 (see Section IVC and E). (91 ). These results demonstrate that levels of various products derived from pro-TRH can be posttranslationally regulated in an independent fashion under altered physiological conditions. Thus, it is logical that neuroendocrine inputs into the PVN can affect pro-TRH processing as well.
Finally, we note that while the genomic organization of the rat prepro-TRH gene is well described (155 156 ), the molecular mechanisms regulating the expression of this gene are incompletely understood. The 5'-region of the prepro-TRH gene contains TATA and GC box sequences, also present in the promoter region of other neuropeptide genes (155 ). In addition, sequences similar to a cAMP response element (CRE), and negative thyroid response elements (TREs), are present. The region between -47 and +6 of the rat prepro-TRH gene is active in CA77 TRH-secreting medullary thyroid carcinoma cells (155 157 ), but not in transgenic mice (158 ). Inclusion of most of exon 1 (bp -47 to +84) increases promoter activity in CA77 cells and activates the promoter in transgenic mice, principally in prepro-TRH gene-producing tissues. Thus, cis element(s) located within exon 1 are necessary for the expression of the rat prepro-TRH gene in vivo (158 ). In CA77 cells, the human prepro-TRH gene is regulated by thyroid hormone through two distinct classes of negative TREs (157 ), similar to other neuropeptide genes such as prepro-SRIF (159 ).
F. Glucocorticoids modulate the biosynthesis and processing of
pro-TRH
Glucocorticoids evoke a broad spectrum of responses in many
eukaryotic cells by stimulating or repressing the transcription of
glucocorticoid-regulated genes, including those of peptide hormones
(160 ). The primary effect of glucocorticoids on gene transcription can
occur by specific binding of the steroid receptor complex to DNA at the
site of glucocorticoid response elements. Glucocorticoids can also
interfere with the action of other transcription factors through
protein-protein interactions and may elicit secondary effects at the
posttranscriptional, translational, and posttranslational levels
(161 162 163 ). For example, glucocorticoids stimulate processing of the
precursors for atrial natriuretic factor and neurotensin (NT) (162 163 ). Glucocorticoids also regulate the posttranslational maturation,
the intracellular trafficking, and the extracellular release of the
mouse mammary tumor virus (164 ).
Glucocorticoids enhance TRH gene expression in several in vitro cell systems, including hypothalamic neurons, anterior pituitary cells, and thyroid C cells, an effect that occurs, at least in part, through transcriptional activation (165 ). Dexamethasone substantially elevates biosynthesis of the 26- kDa TRH prohormone and its intermediate products in cultured anterior pituitary cells, consistent with an overall up-regulation of both the biosynthesis and processing of the TRH precursor (161 ). This explains why glucocorticoids act not only at the transcriptional level, but also at the translational/post-translational level. This question can be addressed in experiments with AtT20 cells transfected with prepro-TRH cDNA driven by a CMV-IE promoter not responsive to physiological signals. Dexamethasone causes a 75% increase in newly synthesized 26-kDa pro-TRH without altering prepro-TRH mRNA levels, suggesting that glucocorticoids raise translation rates and/or slow processing of pro-TRH. In fact, dexamethasone treatment accelerates TRH precursor processing.
Interestingly, processing of the N- vs. the C-terminal
intermediates in the AtT20 cells is influenced
differentially by glucocorticoids. Levels of the N-terminally derived
peptide prepro-TRH25–50 are enhanced while levels of the
5.4-kDa C-terminally derived peptide are reduced. TRH content is
increased (Fig. 7) (161 ). How could
dexamethasone differentially affect the processing of N- vs.
the C-terminal intermediates? Glucocorticoids may alter pro-TRH
processing through changes in the expression of processing enzymes, as
well as morphological alterations in AtT20 cells. For
example, GC volume is obviously enlarged in AtT20 cells
treated with dexamethasone. Although speculative, these changes may
slow down the normal transport of intermediate products from the TGN to
ISGs, thereby altering the accumulation or degradation of intermediate
forms through changes in processing enzyme exposure (84 161 ).
|
In conclusion, glucocorticoids induce changes in the biosynthesis and processing of pro-TRH by affecting both transcription and translation rates, and by differentially influencing the processing of N- vs. C-terminal intermediates of pro-TRH. At the translational and posttranslational level, these effects result in an increase in TRH production, with more complicated differential effects on the accumulation of other N- and C-terminal pro-TRH-derived peptides. It is clear that control over the diverse range of pro-TRH-derived peptides within a specific cell is accomplished mostly from the regulation at the posttranslational level rather than the translational or transcriptional levels. Three examples supporting this hypothesis are presented in this review: 1) pro-TRH processing in the PAG is regulated during the opiate withdrawal, so that levels of TRH remain unchanged, but other pro-TRH-derived peptides are induced (Section II.B.6); 2) pro-TRH processing is regulated during suckling, where a selective, yet dramatic, increase in prepro-TRH178–199 and prepro-TRH186–199 peptides is observed (Section IV.D and E); and 3) in the absence of transcriptional effects, glucocorticoids induce differential processing of pro-TRH in both primary cultures of pituitary cells and transfected AtT20 cells encoding prepro-TRH cDNA (this section).
G. Leptin regulates pro-TRH biosynthesis
Food deprivation in animals and hum
