Age-associated changes in hypothalamic and pituitary neuroendocrine gene expression in the rat.

A cDNA membrane array displaying 1183 probes was used to detect hypothalamic and pituitary changes in gene expression accompanying ageing and age-associated pituitary macroadenomas. Four groups of male Sprague-Dawley rats (3-, 15-, 24-month-old and 24-month-old with prolactinoma) were compared in two independent hybridizations. cDNA array data were confirmed and completed by comparative reverse transcriptase-polymerase chain reaction on selected genes. The expression of 454 and 116 mRNAs was detected in hypothalamus and pituitary, respectively. Growth hormone (GH) mRNA alone represented 85% of total gene expression in the gland of young rats, and other pituitary hormone transcripts 2.8%, while melanin-concentrating hormone (MCH) mRNA, the most expressed neuropeptide transcript involved in neuroendocrine regulation, accounted for only 0.8% of total hypothalamic transcripts. The proportion of genes modified in the hypothalamus and pituitary was rather modest: 1.5% and 5.2%, respectively, for ageing per se, and 1.1% and 5.2% for age-associated macroprolactinomas. Among pituitary specific RNAs, GH mRNA expression was notably decreased with age. At the hypothalamic level, expression of genes directly involved in GH regulation, such as somatostatin and growth hormone-releasing hormone, was not altered, while neuropeptide transcripts involved in feeding behaviour [orexin/hypocretin, MCH, pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART)] were significantly altered. In addition, a few ubiquitous transcripts (hnRNP-K, PFKm, CCND 2, calponin and set) were differently affected in both tissues. Modifications in hypothalamic orexigenic (orexin, MCH) and anorexigenic (POMC, CART) gene expression are in keeping with an age-associated decrease in energy consumption but a higher one in the presence of macroprolactinomas.

Desensitization of type 1 angiotensin II receptor subtypes in the rat kidney.

Differences involving serine residues in the sequence of the carboxyl-terminal tail of type 1 angiotensin II (Ang II) receptor subtypes AT(1A) and AT(1B) suggest differences in desensitization ability. We examined the Ang II-induced homologous desensitization patterns of both receptor subtypes in freshly isolated renal structures: glomerulus (Glom), afferent arteriole, and cortical thick ascending limb (CTAL), whose content in each subtype mRNA is different, by measuring variations in intracellular calcium concentration. A preexposure to a maximal dose of Ang II, followed by a second application of the same concentration, induced: 1) a complete desensitization in Glom, where AT(1A) and AT(1B) mRNAs were expressed in similar proportions, and 2) no or partial desensitization in afferent arteriole and CTAL, where AT(1A) mRNA was predominant. In the absence of nephron structure containing only AT(1B) mRNA, we studied rat anterior pituitary cells that exhibit high content in this subtype and observed that desensitization was not complete. In Glom, CTAL, and pituitary cells, desensitization proceeded in a dose-dependent manner. In Glom and CTAL, desensitization occurred via a PKC-independent mechanism. These results suggest that desensitization does not depend on the nature of Ang II receptor subtype but either on the proportion of each subtype in a given cell and/or on cell specific type. This could allow adaptive biological responses to Ang II appropriate to the specific function of a given cell type.

Differential pituitary gene expression profiles associated- to aging and spontaneous tumors as revealed by rat cDNA expression array.

Aging of the rat pituitary is often accompanied by the occurrence of adenomas. We asked whether complementary DNA hybridization array was adapted to identify gene expression patterns linked to aging and associated spontaneous adenomas. Thus, [32P]dATP-labeled cDNAs were prepared from pituitaries of three month-old rats (Y) and tumor-bearing 20-28-month-old rats (OT). The cDNAs were hybridized to identical membrane arrays allowing to study simultaneously 588 known genes (Clontech 7738-1). Among the 79 genes detected, the GH gene was predominantly expressed in both groups. Twenty-eight genes in the OT group and 15 in the Y group were found to be expressed at a higher level. The largest differences were of about 17 fold and were observed for the galanin and glutathione S transferase genes in the Y and OT groups, respectively. Relative RT-PCR was applied to validate the OT versus Y expression pattern obtained via cDNA array hybridization. The results were consistent for 14 out the 15 genes tested. In the light of these results, differential membrane array hybridization appears suitable to identify gene expression profiles associated with pituitary aging.

Effect of 17beta-estradiol on somatostatin receptor expression and inhibitory effects on growth hormone and prolactin release in rat pituitary cell cultures.

In the present study, we tested whether 17beta-estradiol (E2)-induced PRL sensitivity to somatostatin-14 (SRIF) involves selective up-regulation of discrete somatostatin receptor subtypes (ssts) in primary cultures of female rat pituitary cells. The efficacy of the endogenous peptide SRIF to inhibit GH and PRL secretion and cAMP accumulation was compared with those of octreotide (OCT), BIM-23052, BIM-23056, and BIM-23268, which have been reported to be relatively selective for rat sst2, sst3, and sst5. Experiments were performed in steroid-depleted media supplemented or not with 1 nM E2 for 96 h. SRIF, OCT, and BIM-23052 inhibited cAMP accumulation and GH release independently of E2. In contrast, all three agonists affected PRL release in E2-treated cultures only. Inhibition of cAMP accumulation by SRIF, OCT, and BIM-23052 was enhanced by exposure of cells to E2. The rank of potency of the agonists, OCT = SRIF > BIM-23052, was similar for GH and PRL inhibition. BIM-23268 was a weak agonist on GH, but not on PRL, secretion. BIM-23056 had no effect on the release of either hormone, but slightly inhibited cAMP formation in E2-treated cells. To verify whether SRIF receptor gene expression correlated with these observations, messenger RNA (mRNA) transcripts corresponding to the five ssts were measured by quantitative RT-PCR in the presence or absence of E2. Control cells expressed predominantly sst2 and sst3 transcripts; sst1 mRNA was present in moderate amounts, whereas sst4 and sst5 were only weakly expressed. E2 had a differential effect on distinct ssts; it increased mRNA concentrations corresponding to sst2 and sst3, and decreased that of sst1. These results indicate that sst2 and sst3 receptors are the major somatostatin receptors expressed in the female rat pituitary, and that both of them are positively regulated by estradiol. However, the capacity of lactotropes to respond to SRIF after exposure to E2 seems to depend more upon E2-induced up-regulation of the sst2 than of the sst3 receptor subtype.

Hypothalamic and hypophyseal regulation of growth hormone secretion.

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice.

The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the beta-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHbeta gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.

Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

CpG methylation represses the activity of the rat prolactin promoter in rat GH3 pituitary cell lines.

In the present report, we have investigated the role of DNA methylation on the binding and trans-acting properties of transcription factors involved in the regulation of the rat prolactin (rPRL) gene, specifically Pit-1. To this aim we took advantage of a model system composed of three GH3 rat pituitary tumor cell lines that greatly differed in the extent of rPRL gene methylation and in the level of rPRL gene expression. Northern blot analyses indicated that identical species of Pit-1 mRNA were present to similar extent in the three GH3 cell lines. Electrophoretic mobility shift assays further demonstrated that Pit-1 was present in nuclear extracts and displayed equal affinities to bind the 1P responsive element encompassing the -65 to -38 region of the rPRL promoter, whatever the GH3 cell line tested. These data suggested that differential expression of the rPRL gene among cell lines did not result from variable amounts of Pit-1. By combining in vitro methylation and transient transfection experiments with a rPRL promoter-driven CAT construct, we showed that extensive methylation at CpG sites abolished the expression of the reporter gene. Furthermore, in vivo competition assays demonstrated that CpG methylation inhibited gene expression by preventing the binding of transcription factors We propose that related mechanisms linked to DNA methylation might alter the activity of the endogenous PRL gene in the low expressing cell line.

Multiple intracellular signallings are involved in thyrotropin-releasing hormone (TRH)-induced c-fos and jun B mRNA levels in clonal prolactin cells.

In mammosomatotropes GH3B6 cells, one of the primary responses to thyrotropin-releasing hormone (TRH) is the parallel induction of two proto-oncogenes, c-fos and jun B, which code for constituents of AP1 transcription factor. To better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signals in the induction of each proto-oncogene on the one hand, and on prolactin (PRL) release and PRL gene expression on the other hand. Northern and dot-blot analyses revealed that the activation of protein kinase C (PKC)-, Ca(2+)- or adenylyl cyclase-dependent pathways acutely increased both c-fos and jun B transcripts. However, a gene specific responsiveness was revealed using phorbol 12-myristate 13-acetate (TPA) and several combined treatments. The simultaneous activation of PKC and Ca(2+)-dependent pathways resulted in synergistic stimulations of c-fos mRNA levels only. Consistently, ionomycin plus low doses of TPA solely reproduced the potent effect of TRH on c-fos transcripts. Data collected from TRH and TPA down-regulated cells indicated that TRH probably recruits TPA-dependent PKC isoforms for stimulating c-fos but not jun B transcripts. On the contrary, the TRH-induced stimulation of either proto-oncogene likely involves Ca(2+)-dependent mechanisms because calcium agonists and the peptide exert non-additive effects. Finally, the synergistic stimulations observed in response to TRH combined with forskolin, indicate that adenylyl cyclase-dependent mechanisms are interconnected with TRH-induced proto-oncogene expression. The overall study also reveals that among the agonists tested, the dihydropyridine Bay K 8644 and forskolin only were capable to induce a long-lasting stimulation of c-fos and jun B mRNA levels, concomitant to increased levels of PRL transcripts, as does TRH. Considering that AP1 is assumed to be involved in signal transmission from the cell surface to the nucleus, it might be thus proposed that a common stimulation of c-fos and jun B gene expression is possibly involved in the activation of the PRL gene. On the other hand, the systematic coincidence between acute PRL release and proto-oncogenes expression suggest a role for c-fos and jun B in the control of genes involved in the secretory process.

[Stimulation of C-fos and jun B proto-oncogenes: potential role of TRH effects in clone cell line with prolactin (GH3B6)]. / La stimulation des proto-oncogènes c-fos et jun B: un relais potential des effets du TRH dans une lignée clonale de cellules à prolactine (GH3B6).

The hypothalamic neuropeptide TRH, which stimulates prolactin (PRL) release and PRL gene transcription, also raises c-fos proto-oncogene mRNA levels in GH3B6 rat pituitary cells. C-fos is assumed to be involved in the transduction of external signals to the nucleus as a component of AP1 transcription factor, a protein complex that contains a member of the jun proto-oncogene family. We have thus looked for the member(s) of the jun family that could be the partner of c-fos in TRH-stimulated GH3B6 cells. The common biphasic pattern of jun B and c-fos mRNA regulation under TRH exposure, i.e., an early peak and a long-lasting plateau phase, suggested that jun B was the best candidate. Then, to better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signalings in the induction of each proto-oncogene. This was done taking as a model that the effects of TRH on PRL release and PRL gene transcription has been previously ascribed to the coupling of the TRH receptor to the activation of both protein kinase C- and calcium-dependent mechanisms. An extensive pharmacological analyses revealed that PKC-, Ca2+ but also protein kinase A-dependent mechanisms are involved in TRH-induced c-fos and jun B mRNA early responses in GH3B6 cells. The overall study also revealed specific features in the control by TRH of each proto-oncogene by some intracellular messengers. Finally, considering the fact that second long lasting phase of proto-oncogene expression was found associated with increased PRL mRNA accumulation whatever the stimulus, it might be proposed that AP1 [c-Fos/Jun B] factor could be involved in the regulation of PRL gene expression. Such hypothesis was furthermore supported by preliminary gel-shift experiments. Nevertheless, in view of the systematic coincidence between acute PRL release and early proto-oncogene induction, a role for c-fos and jun B in the control of genes involved in the secretory process might also be suggested.

Production and characterization of polyclonal anti-idiotypic anti-TRH antibodies: application to the study of pituitary TRH receptor.

cDNA encoding the thyrotropin-releasing hormone receptor (TRH-R) was recently cloned in rat pituitary prolactin cells and in mouse thyrotropes. The molecular weights of the protein sequences obtained are 46.6 and 44.5 kD. However, TRH-R has not yet been purified to homogeneity and specific anti-TRH-R antibody could not yet be obtained by classical biochemical methods. We thus attempted to obtain antibodies specific for TRH-R using an anti-idiotypic approach. Rabbits of the same allotype were immunized using Igs (Ab1) extracted from rabbit polyclonal anti-TRH immune serum. Anti-idiotypic rabbit polyclonal anti-anti-TRH antibodies (Ab2) were obtained, as shown by their ability to inhibit the formation of TRH-anti-TRH complexes in a radioimmunoassay system. One of them, the polyclonal Ab2 R38/B12, was tested for its ability to recognize the TRH-R in rat pituitary, tumor-derived, GH3/B6 prolactin-secreting cells. Immunoreactive material was immunocytochemically detected in fixed and saponin-permeabilized GH3/B6 cells. The immunostaining was localized at the plasma membrane and on intracellular structures. It was not observed using non-anti-TRH Ab2 and was abolished in the presence of excess TRH. Furthermore, binding of [125I]R38/B12 on fixed and saponin-permeabilized GH3/B6 cells was partially inhibited by excess TRH. By immunoblot analyses of Triton X-114 cell extracts performed under reducing or nonreducing conditions, the polyclonal R38/B12 Igs revealed two main protein species of approximately 98 and approximately 76 kD as well as several proteins < or = 46 kD. In the presence of excess TRH, the approximately 98- and approximately 42-kD bands were abolished, whereas the intensity of the other bands was faintly attenuated only. The approximately 98-kD protein was also revealed in a two-dimensional PAGE analysis. Nevertheless, the effects of R38/B12 Igs on [3H]TRH binding by GH3/B6 cells and on basal or TRH-induced prolactin secretion were not markedly different from those elicited by control Ab2. These data suggest that we have characterized Ab2 antibodies which recognize a molecular entity that might be related to the TRH-R in GH3B6 cells.

Thyrotropin-releasing hormone stimulates in parallel jun B and c-fos messenger ribonucleic acids in GH3B6 pituitary cells: comparison with PRL secretion.

The hypothalamic neuropeptide TRH stimulates PRL release and PRL gene transcription in GH3B6 rat pituitary cells. In this model, TRH also raises c-fos proto-oncogene mRNA levels. c-fos is assumed to be involved in the transduction of external signals to the nucleus as a component of AP1 transcription factor, a complex that contains a member of the jun proto-oncogene family. Hence, the main aim of this study was to look for the expression of c-jun, jun B, and jun D proto-oncogenes in TRH-stimulated GH3B6 cells, compared to c-fos mRNA levels and PRL secretion. This was performed using serum-starved cells. Northern blotting and dot blotting revealed that jun B mRNA were expressed at noticeable levels in control cells and were strongly stimulated by TRH. This stimulation was dose-dependent and exhibited an early peak and a long-lasting plateau phase stabilized at 4 h, similar to TRH-induced c-fos mRNA induction. c-jun mRNA was expressed at minute levels in control cells and was transiently stimulated by TRH. The jun D message was not detected. TRH-induced jun B and c-fos responses were characteristic of immediate early genes as shown by the superinduction observed under cycloheximide treatment and the total inhibition elicited by actinomycin D. Finally, the responses to serum at 30 min and 4 h revealed differences among the proto-oncogenes studied and indicate that the action of TRH is specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic growth hormone (GH) hypersecretion induces reciprocal and reversible changes in mRNA levels from hypothalamic GH-releasing hormone and somatostatin neurons in the rat.

Effects of growth hormone (GH) hypersecretion on somatostatin-(SRIH) and GH-releasing hormone (GHRH) were studied by in situ hybridization and receptor autoradiography in rats bearing a GH-secreting tumor. 6 and 18 wk after tumor induction, animals displayed a sharp increase in body weight and GH plasma levels; pituitary GH content was reduced by 47 and 55%, while that of prolactin and thyrotropin was unchanged. At 18 wk, hypothalamic GHRH and SRIH levels had fallen by 84 and 52%, respectively. In parallel, the density of GHRH mRNA per arcuate neuron was reduced by 52 and 50% at 6 and 18 wk, while SRIH mRNA levels increased by 71 and 83% in the periventricular nucleus (with no alteration in the hilus of the dentate gyrus). The numbers of GHRH- and SRIH-synthetizing neurons in the hypothalamus were not altered in GH-hypersecreting rats. Resection of the tumor restored hypothalamic GHRH and SRIH mRNAs to control levels. GH hypersecretion did not modify 125I-SRIH binding sites on GHRH neurons. Thus, chronic GH hypersecretion affects the expression of the genes encoding for GHRH and SRIH. The effect is long lasting, not desensitizable and reversible.

Properties of monoclonal antibodies to thyroliberin (TRH) induced by different immunogens: comparison with pituitary TRH receptor.

Thyroliberin E-H-P-NH2 (TRH) is a small neuropeptide pGlu-His-Pro-NH2 widely distributed in neural sites. The aim of this work was to obtain an antibody molecule with the nearest properties to that of TRH-receptor in GH3 cells. Different TRH-protein conjugates were prepared and utilized to induce monoclonal antibodies in mice. Several monoclonal antibodies were obtained using E-H-P-NH2 (TRH) coupled either to the histidyl residue (immunogen I) or to the prolyl residue (immunogen II). Antibodies generated using immunogen I and immunogen II were characterized in a radioimmunoassay system and an enzyme immunoassay system respectively. Their selectivities regarding a series of TRH related peptides were compared to those of rabbit polyclonal antibodies using three differently labelled TRH (tritiated-TRH, mono-iodinated-TRH and TRH-OH-acetyl-cholinesterase) as tracers and to prolactin secreting cells TRH receptors using 3H-TRH. Whatever the immunogen, the stereospecificity of monoclonal antibodies tested were found more different from TRH receptor characteristics than rabbit polyclonal antibodies.

Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells.

Secretogranin I (SgI; chromogranin B) belongs to a class of acidic tyrosine-sulfated secretory proteins believed to play a role in the secretory process of endocrine cells. Our aim here was to compare the levels of SgI mRNA to that of prolactin (PRL) and growth hormone (GH), using rat pituitary cell lines. As far as the constitutive expression is concerned, we found a positive correlation between SgI mRNA and PRL mRNA levels. However, the neuropeptide TRH (50 nM) inhibited the accumulation of SgI mRNA in GH3B6 cells whereas, as expected, it induced a rapid and sustained increase in PRL mRNA accumulation. By contrast, 17 beta-estradiol (1 nM) stimulated the accumulation of both SgI and PRL mRNAs, with the same EC50 (18-59 pM). Reciprocally, treatment with dexamethasone (100 nM) reduced the level of SgI and PRL mRNAs to 23% and 29% of control, respectively, but led to a 2.1-fold increase in the GH mRNA level. Altogether, the present work shows that SgI gene expression is subject to multiple hormonal regulations and occasionally parallels the regulation of the PRL gene but never that of the GH gene, under the conditions tested.

[Neuropeptides and regulation of various proto-oncogenes in cultured anterior pituitary cells]. / Neuropeptides et régulation de certains proto-oncogènes dans les cellules antéhypophysaires en culture.

The analysis of the characteristics of a number of proto-oncogenes reveals some similarities with the site of action (plasma membrane receptor, nucleus) or the mechanisms of action (coupling protein of the "G" family, transcriptional activity) of neuropeptides and namely hypophysiotropic hypothalamic neuropeptides. The example illustrated in this review concerns the induction of the early nuclear oncogene c. fos by TRH in a prolactin secreting rat pituitary cell line. TRH regulates both, hormone release and biosynthesis, in this cell line. Kinetic and pharmacological studies suggest that the induction of the proto-oncogene mRNA accumulation is rather associated with the TRH-induced release of the stored hormone than with the increased transcriptional activity of the prolactin gene.