Rat Pituitary Tumor Cell Line Research Articles

Local action of estrogen and thyroid hormone on vasoactive intestinal peptide (VIP) and galanin gene expression in the ratanterior pituitary

Neuropeptides act within the pituitary as autocrine or paracrine factors, modulating the synthesis and release of primary pituitary hormones, and possibly regulating cell proliferation and/or plasticity. Manipulation of the endocrine status of rats produces dramatic long-term changes in the pituitary expression of several peptides, including the neuropeptides galanin and vasoactive intestinal peptide (VIP). Whether or not these changes are caused indirectly by hypothalamic factors, or by hormone actions directly in the pituitary, has been only partially addressed. To determine if estrogen or thyroid hormone can act directly within the pituitary to regulate VIP and galanin gene expression, cultured female rat pituitary cells were treated with 10nM 1,17 β-estradiol (E2) or triiodothyronine (T3). E2treatment for three days resulted in an approximate 5-fold and 7-fold increase in VIP and galanin mRNA, respectively. In contrast, T3 treatment reduced the mRNA levels of these neuropeptides to approximately 40% and 30% of control values. A time course study indicated that the actions of estrogen on VIP and galanin mRNA, and of thyroid hormone on VIP mRNA were readily apparent after 24h.The rat pituitary tumor cell line RC-4B/C was found to express easily detectable levels of galanin but not VIP mRNA. Galanin gene expression in these cells was moderately increased by E2 and decreased by T3. Transfection of a series of luciferase plasmids containing 5kb to 131bp of the bovine galanin promoter fused to luciferase revealed cell-type specific enhancer sequences located between −452 and −131bp of the galanin gene transcription start site. However, transfected plasmids were minimally responsive to E2 and T3 treatment. Overall the results suggest that E2 and T3 exert significant local actions in the pituitary on VIP and galanin gene expression. The bovine galanin gene fragment used in these studies contains a potential pituitary cell-type specific enhancer, but appears to lack strong E2-and T3-responsive sequences.

Insulin receptor tyrosine kinase activity and phosphorylation of tyrosines 1162 and 1163 are required for insulin-increased prolactin gene expression

Insulin treatment increased prolactin gene expression in GH4 cells, a rat pituitary tumor cell line, through the endogenous insulin receptor. However, insulin regulation of transfected plasmids required the expression of cotransfected insulin receptor. Prolactin-CAT expression was increased 12-fold in cells transfected with wild type insulin receptor, but insulin did not increase prolactin gene expression when a kinase negative mutant of the ATP binding site (K1030R) was expressed. Thus, receptor kinase activity was required for signaling to gene transcription. Mutation of tyrosine 1158 did not reduce insulin-increased prolactin-CAT expression while individual mutations of tyrosine 1162 and tyrosine 1163 each reduced insulin-increased prolactin-CAT expression by 50% and a triple mutant of tyrosines 1158/1162/1163 was inactive. Thus, mutation of tyrosine 1162 and 1163 was also sufficient to inactivate signaling by the insulin receptor. Insulin-stimulated auto phosphorylation occurred in all mutants in vitro except the ATP binding site mutant. However, the ability of mutant insulin receptors to mediate insulin-increased prolactin-CAT expression correlated with the substrate-specific catalytic activity of the receptors. This suggested that phosphorylation of these tyrosines was important for substrate access to the catalytic domain of the receptor.

Strain difference in regulation of pituitary tumor transforming gene (PTTG) in estrogen-induced pituitary tumorigenesis in rats.

Recently a novel oncogene, PTTG (pituitary tumor transforming gene) was isolated from a rat pituitary tumor cell line whose expression is apparently correlated with pituitary tumorigenesis. In the rat, estradiol (E2) is known to induce anterior pituitary hyperplasia. The effects of E2, however, vary greatly among rat strains. Therefore we examined the expression of PTTG and its regulation by E2 in F344, Wistar, Brown–Norway and Donryu rats. Four–week–old females were ovariecto–mized and a pellet containing 10 mg of E2 was given s.c. Total RNA was isolated from the pituitary gland and PTTG mRNA was measured with a competitive RT–PCR technique. The F344 strain was the most susceptible to E2 induction of pituitary tumorigenesis, followed by Wistar and Brown–Norway, while no increase in pituitary weight was noted in Donryu rats. PTTG mRNA in the gland was induced by E2 within 48–72 h in F344 and Wistar, but not in Brown–Norway or Donryu strains. These data suggest that PTTG expression may at least in part be responsible for strain differences in E2–induced pituitary tumorigenesis

Dexamethasone blocks antioestrogen- and oxidant-induced death of pituitary tumour cells.

The oestrogen receptor is fundamental to the growth and survival of the rat pituitary tumour cell line, GH(3). Our previous studies have shown that antioestrogens such as RU 58668 and ZM 182780 will reduce the rate of cell division and also induce cell death. Death of these cells in response to antioestrogen treatment appears to be due to a heightened sensitivity to reactive oxygen species (ROS). As part of a study to determine the cross-talk between steroid receptor systems in these cells, we have observed that the glucocorticoid, dexamethasone (Dex), inhibits antioestrogen-induced cell death. Cell death induced by H(2)O(2) is enhanced by ZM 182780 and this effect is also blocked by Dex. As apoptotic cell death in a number of systems involves an early loss of mitochondrial membrane potential (DeltaPsi(m)), we have performed detailed studies on the time-course of DeltaPsi(m) loss in relation to the loss in cell membrane function. These studies have indicated that a loss of DeltaPsi(m) parallels a loss of cell membrane function - this is more characteristic of necrosis than of apoptosis. From microscopic observations of these cells in response to H(2)O(2), it has been noted that early cell membrane blebbing, induced by H(2)O(2), is blocked in the presence of ZM 182780. Cell membrane blebbing can precede necrosis as well as apoptosis and it is thought to involve cytoskeletal changes, for which localised glycolytic reactions provide ATP. These observations, together with those showing that removal of glucose, but not inhibition of mitochondrial function, enhances ROS-induced cell death, prompted studies on the glycolytic pathway. As a strong candidate mechanism, it would appear that, via an effect on one of the rate-limiting glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, Dex is able to overcome the antioestrogen-enhanced loss of glycolytic function following exposure of cells to ROS. This report contributes to the growing body of evidence showing that glucocorticoids provide a survival advantage to both normal and tumour cell types.

Metabolic Activation of the Proestrogens trans-Stilbene and trans-Stilbene Oxide by Rat Liver Microsomes

A liver microsome-mediated activation of the proestrogens trans-stilbene and trans-stilbene oxide was found in this study. trans-Stilbene and trans-stilbene oxide were negative in estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7 and growth assay in rat pituitary tumor cell line MtT/E-2. However, these compounds exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. In contrast, cis-stilbene and cis-stilbene oxide did not show estrogenic activity after such incubation. When trans-stilbene was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, two metabolites were detected by HPLC. They were identified unequivocally as trans-4-hydroxystilbene and trans-4,4′-dihydroxystilbene by mass and UV spectral comparison with authentic samples. The oxidase activity of the liver microsomes toward trans-stilbene was inhibited by SKF 525-A and α -naphthoflavone. Minor activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. trans-4-Hydroxystilbene and trans-4,4′-dihydroxystilbene exhibited significant estrogenic activities. These results suggest that the estrogenic activities of trans-stilbene and trans-stilbene oxide were due to formation of hydroxylated metabolites.

Characterization and distribution of extracellular matrix components and receptors in GH 3B 6 prolactin cells

GH 3B 6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50–70 % of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH 3B 6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: α5β1 (FN receptor), α6β1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75 %) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH 3B 6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Overexpression of vascular endothelial growth factor164 and its co-receptor neuropilin-1 in estrogen-induced rat pituitary tumors and GH3 rat pituitary tumor cells.

We have shown previously that the VEGF system plays a crucial role in regulation of tumor angiogenesis during the development of estrogen-induced prolactin-secreting pituitary tumors in Fisher 344 rats. Studies also suggested that both endothelial and non-endothelial cells expressed VEGF. However, several questions concerning the VEGF signals in regulation of estrogen-induced angiogenesis in rat pituitary remained unanswered. VEGF exists in a number of isoforms in human and rodent tissue (i.e., VEGF206h/205r, VEGF189h/188r, VEGF165h/164r, VEGF145h/144r and VEGF121) that differ in their molecular masses and biological activities. The VEGF isoforms bind with two tyrosine-kinase receptors, KDR/flk-1 and flt-1. In addition, VEGF165 binds with a newly identified co-receptor, neuropilin-1, which is expressed in human endothelial cells and several types of non-endothelial cells including tumor cells. The present study was undertaken to elucidate which isoforms of VEGF are predominantly expressed in normal Fisher 344 rat pituitaries, estrogen-induced prolactin secreting rat pituitary tumors and in prolactin secreting rat pituitary tumor cell line (GH3 cell line). To identify the isoform, RT-PCR with primer pairs derived from exon 1 and exon 8 of the VEGF gene, cloning, sequencing and Western blot analysis were performed. The status of neuropilin-1 in the rat pituitaries (normal and transformed) and GH3 pituitary tumor cell line has also been investigated using RT-PCR and Western blot analysis. These studies demonstrate that normal rat pituitaries, estrogen-induced rat pituitary tumors and GH3 pituitary tumor cells expressed VEGF164 and co-receptor, neuropilin-1. The VEGF164 was the predominant form in all of these cells. The VEGF164 and neuropilin-1 mRNA and protein levels were significantly higher in the estrogen-induced pituitary tumors and GH3 tumor cell line, as compared to normal pituitary. The data suggest that both VEGF164 and neuropilin-1 may actively participate in modulation of tumor angiogenesis and the development of pituitary tumors in Fisher 344 rats.

Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and responses to oestradiol and xenoestrogens.

Our laboratory has identified plasma membrane oestrogen receptors on a GH3/B6 rat pituitary tumour cell line and several sublines which produce rapid (within minutes), non-genomic responses to oestrogens. Oestrogen receptors have been identified by their binding to nine different antibodies (Abs) which together recognize at least seven epitopes on the oestrogen receptor-alpha. GH3/B6/F10 cells, a membrane oestrogen receptor-enriched subline, elevate intracellular calcium levels in response to 10 nM oestradiol. Prolactin release in these cells is triggered by both 1 pM and 1 nM oestradiol and diethylstilbestrol (DES). A membrane oestrogen receptor-alpha immunocytochemical signal rapidly disappears (at 3 min) and reappears (at 12-15 min) when 1 nM oestradiol, 10 nM diethylstilbestrol, or 10 nM nonylphenol is applied to the cells. This suggests that both oestrogens and xenoestrogens can utilize this alternative pathway for oestrogenic action. Xenoestrogens, which have so far shown weak effects in genomic assay systems, should now be retested for activity in eliciting membrane-initiated oestrogenic responses.

Calcium-dependent inactivation of neuronal calcium channel currents is independent of calcineurin

Dephosphorylation by the Ca 2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca 2+-dependent inactivation of voltage-gated Ca 2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca 2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca 2+ channels was studied in a rat pituitary tumor cell line (GH 3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH 3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca 2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca 2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba 2+ substituted for extracellular Ca 2+ or when the Ca 2+ chelator EGTA was present intracellularly, indicating that both types of Ca 2+ currents exhibited Ca 2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca 2+ channel current inactivation of either L- or N-type Ca 2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca 2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca 2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.

An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family.

A novel proto-oncogene, PTTG (Pituitary Tumor Transforming Gene), was isolated in our laboratory by virtue of its increased expression in rat pituitary tumor cell lines. Cells which overexpress human or rat PTTG form tumors in athymic mice. hPTTG is highly expressed in cancer cell lines, pituitary adenomas and in normal testis, suggesting that hPTTG protein has different tissue-specific interactions in normal cells and in cancer. Alternatively, different hPTTG gene family members may be functional in normal development and in tumorigenesis. While mapping the chromosomal location of hPTTG to 5q33, we discovered a second gene, hPTTG2, which is intronless and maps to chromosome 4p12. Using gene-specific oligonucleotide hybridization in a PCR-ELISA assay, we determined that hPTTG2 is expressed in both normal and tumorous pituitary. However, high levels of hPTTG mRNA in cancer cell lines are due to increased expression of hPTTG1. Thus, this family of proto-oncogenes appears to differentially participate in tumor-specific pathogenesis.

Establishment of an estrogen responsive rat pituitary cell sub-line MtT/E-2.

A rat pituitary tumor sub-line MtT/E-2 was established from a rat pituitary tumor cell line MtT/E. Its growth was found to depend on the presence of estradiol (E2) in culture media at 10(-13)-10(-9) M, whereas the original cell line MtT/E proliferated autonomously. The recently discovered PTTG (pituitary tumor transforming gene) is highly expressed in this cell line, although not regulated by E2. On the other hand, E2 induced c-myc and cyclin D1 proteins in MtT/E-2, which contains a lot (220+/-18 fmol/mg protein) of estrogen receptor demonstrated by RT-PCR analysis to be predominantly alpha type. MtT/E-2 secretes growth hormone which is, interestingly, regulated by retinoic acid and dexamethasone rather than thyroid hormones.

The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment with 17beta-estradiol, insulin and EGF.

The mechanisms for the formation of autophagic vacuoles were investigated using GH4C1 cells, a rat pituitary tumor cell line, whose induction increases intracellular levels of lysosomal proteinases and their mRNA by treatment with a combination of hormones (17beta-estradiol, insulin and EGF). By ordinary electron microscopy, autophagic vacuoles containing various undigested structures with or without limiting membranes were abundant in the hormone-induced cells. These vacuoles, also containing numerous small vesicles, appeared to be derived from multivesicular bodies. In fact, there were also numerous C-shaped multivesicular bodies which enclosed cytoplasmic portions, suggesting that these unique structures are involved in the production of the autophagic vacuoles. Moreover, the cytoplasmic portions enlapped by the C-shaped multivesicular bodies were high in electron density and contained filamentous structures. By the cryothin-section immunogold method, the C-shaped multivesicular bodies in some cases contained lysosomal marker proteins such as cathepsins B and H, and Igp 120. Using an anti-actin monoclonal antibody, immunogold particles clearly labeled the cytoplasmic portions enclosed by the C-shaped multivesicular bodies. Pulse-chase experiments with horse radish peroxidase, a fluid-phase endocytic marker, revealed that the incidence of the C-shaped multivesicular bodies labeled with horse radish peroxidase peaked at 30 min after the beginning of chase incubation, whereas no C-shaped multivesicular body with horse radish peroxidase was detected in the cells by cytochalasin D treatment. These results suggest that the C-shaped multivesicular bodies occur in a transitional process from endosomes to lysosomes by the action of actin filaments, and that this morphological change may be essential for the production of autophagic vacuoles in the hormone-induced GH4C1 cells.

Microcapsules prepared from alginate and a photosensitive poly(L-lysine)

A photosensitive polymer, α-phenylcinnamylideneacetylated poly(L-lysine), was synthesized and characterized. This photosensitive poly(L-lysine) had 10% of its lysine residues reacted with α-phenylcinnamylidene acetyl group and displayed an absorption maximum at 329 nm. The photosensitive poly(L-lysine) was used for the preparation of microcapsules. The capsules formed from this photosensitive poly(L-lysine) and alginate were strengthened significantly by light irradiation. The photo cross-linked capsular membrane was permeable to proteins with mass transfer rate in the descending order: cytochrome C, myoglobin, and serum albumin. GH3 (a rat pituitary tumor cell line) cells were encapsulated and cultured with this microencapsulation system. The cells proliferated to a density of about 4 x 105 cells ml-1 in the capsules after 6 days cultivation.

Presence of growth hormone secretagogue receptor messenger ribonucleic acid in human pituitary tumors and rat GH3 cells.

A novel G11-protein-coupled receptor specific for synthetic GH-releasing peptides (GHRPs) has recently been cloned and sequenced. Two forms exist, types 1a and 1b, the latter of which is biologically inactive. Using RT-PCR, we looked for the presence in tumorous pituitary cells of messenger ribonucleic acid (mRNA) for this novel GH secretagogue receptor (GHS-R). Both subtypes of GHS-R mRNA were detected in all six human pituitary somatotropinomas removed from patients with acromegaly. In culture, four of the tumors exhibited strong responses to GHRP-2 in terms of both phosphatidylinositol (PI) hydrolysis and GH secretion, but two were resistant. There was no apparent difference in the type 1a and type 1b expression pattern, as judged by RT-PCR, between responsive and nonresponsive tumors. Similarly, the rat pituitary tumor cell line, GH3, was found to express GHS-R mRNA, although these cells also did not respond to GHRPs. RT-PCR failed to detect GHS-R mRNA in eight functionless human pituitary tumors. In contrast, prolactinomas were found to express the receptor and, in culture, significant stimulation of PRL secretion and PI hydrolysis occurred in two of three tumors tested. These results demonstrate that tumorous somatotrophs express the GHS-R gene and that the occasionally observed nonresponsiveness of somatotropinomas to GHRPs is not due to the absence of the biologically active type 1a receptor. Additionally, human pituitary prolactinomas also express GHS-R and are able to respond to GHRPs in terms of PI hydrolysis and PRL secretion. In contrast, GHS-R gene expression does not appear to be associated with human functionless pituitary tumors.

Production of uniformly stable isotope labelled proteins from mammalian cells

In order to implement a new strategy for determining the kinetics and metabolism of therapeutic proteins, we had to produce milligram quantities of a uniformly stable isotope labelled species. To produce the labelled protein, the rat pituitary tumor cell line GH3 was cultured in Celtone-M, a commercially prepared medium that contains all the necessary 13C,15N-labelled amino acids as well as a uniformly 13C-labelled carbohydrate source to produce uniformly 13C,15N-labelled rat growth hormone. The growth hormone secreted into the culture medium was purified using ultrafiltration followed by reversed-phase chromatography. The yield of labelled protein was approximately 10 mg/L. SDS-PAGE and western blot analyses confirmed that the labelled growth hormone migrated similarly to the unlabelled material, and that both labelled and unlabelled growth hormone were immunoreactive. Electrospray ionization mass spectrometry (ESI) of the purified material revealed that the molecular weight of the labelled material was 995 units higher (22804 Da) than its unlabelled counterpart (21809 Da). This indicated labelling at 81% of all possible carbon and nitrogen sites throughout the protein. An alternative strategy using HPLC/CRIMS indicated that the protein was 89% 13C-labelled. It is possible to quantitate this stable-isotope labelled protein following iv injection in a rat, using HPLC/CRIMS. These results show that once cells have been adapted to growth in Celtone-M, the preparation of highly and uniformly labelled proteins from mammalian cells appears no more difficult than production of their unlabelled counterparts. This will make a much wider range of proteins available for NMR and other applications that employ stable-isotope labelled proteins. © 1997 John Wiley & Sons, Ltd.

Effect of bromocriptine on the sensitivity of pituitary tumor cells to radiation.

GH3 cells, a PRL-secreting rat pituitary tumor cell line, are sensitive to bromocriptine, and provide a useful model for the present studies. GH3 cells, in the presence or absence of 2.5 x 10(-5) M bromocriptine, were irradiated at doses ranging from 0 to 10 Gy. After irradiation the bromocriptine was washed out, cells were seeded at a density of 10,000 cells/ml, and growth curves were obtained by counting the cell number at weekly intervals for 1 month. Cell growth was inhibited by the radiation in a dose-dependent fashion, with complete inhibition of cell growth by 9 Gy. However, no protection against radiation damage was afforded by bromocriptine. These results might suggest that the combined therapies of tumor shrinkage by bromocriptine and stereotactic irradiation of the sella might be a useful clinical therapeutic strategy, particularly in patients with large tumors, severe hyperprolactinemia, and pre-existing hypopituitarism.

Anin VitroTest System for Thyroid Hormone Action

Anin vitrobioassay for the biological action of thyroid hormones has been developed. The test is based on a dose-dependent increase in growth rate and cell activation of the rat pituitary tumor cell line GH3. This cell line is dependent on thyroid hormones for cell division when plated at low density in serum-free medium. The measurement of the cell-stimulating effect ofL-triiodothyronine (T3) is based on the capacity of mitochondrial enzymes of viable cells to transform the MTT tetrazolium salt into MTT formazan. A dose-dependent response of the MTT signal can be observed within a range from 0.075 to 1 nMT3in the culture medium. The test is performed in 96-well microplates and allows screening of compounds which may interfere with the action of thyroid hormones.

Paracrine stimulation of cell growth by cholecystokinin/gastrin through cholecystokinin-B receptor on GH3 cells in vitro.

Cholecystokinin (CCK) is detected in pituitary tumors but its role remains unknown. On the hypothesis that CCK may facilitate the cell growth in pituitary tumors, we have examined the effect of CCK on cell growth using a rat pituitary tumor cell line, GH3, cultured in a serum-free, chemically defined medium. Addition of sulfated CCK-(26-33) (CCK-8) in two different concentrations (0.5 approximately 1 nM) caused a significant increase in the number of GH3 cells. The antagonist (1 microM) for CCK-B receptor, but not CCK-A receptor, significantly inhibited the number of GH3 cells. Northern blot analysis revealed a significant expression of CCK-B receptor mRNA in GH3 cells, but not in normal rat pituitary glands. In addition, immunoreactive CCK/gastrin was detected by RIA in the GH3 cell extracts as well as the serum-free culture medium. In GH3 cell extracts, both CCK-8 and gastrin like peptides were identified by gel chromatography. These findings provided the first evidence for an autocrine/paracrine role of CCK and gastrin on stimulation of GH3 cell growth through the CCK-B receptor.

Novel location and function of a thyroid hormone response element.

We describe a novel thyroid hormone response element (TRE)-containing sequence, clone 144, isolated by immunoprecipitation of nuclear thyroid hormone receptor (TR)-DNA complexes from the rat pituitary tumor cell line GH4. These cells express several mRNAs of approximately 10 kb that hybridize to the TRE-containing genomic clone 144. These mRNAs are up-regulated at the transcriptional level in the absence of thyroid hormone (T3) and repressed in its presence. The sequence protected from DNase I digestion by TR in clone 144 contains two consensus TRE half-sites arranged as inverted palindromes. The clone 144 TRE is located in the 3' untranslated region (UTR) of several related mRNAs. A reporter construct transfected into 293 cells was responsive to TR regulation when the clone 144 TRE was inserted in the 3' UTR but not when inserted upstream of the promoter. As found for the endogenous 144 mRNAs, the 144 TRE reporter construct is activated by TR in the absence of T3, but not in its presence. Deletion analysis showed that clone 144 sequences flanking the TRE were necessary for TR-mediated regulation, suggesting that the mechanism by which TR regulates transcription through a TRE in the 3' UTR is different from that through the TREs located in the promoter region.

Involvement of interleukin-1 and interleukin-1 receptor antagonist in rat pituitary cell growth regulation.

Interleukin-1 (IL-1), one of the mediators of the interaction between the immune and the neuroendocrine system, is well known to modulate anterior pituitary hormone secretion. As IL-1 influences growth of various cell types, we investigated whether IL-1 is also a growth factor of pituitary cells. We demonstrate that IL-1 dose and time dependently inhibits the growth of normal rat pituitary cells under serum-free conditions. The inhibitory potencies of IL-1 alpha (ED50, 8 pg/ml) and IL-1 beta (ED50, 6 pg/ml) were nearly identical. In the presence of low amounts of serum, both IL-1 alpha (ED50, 130 pg/ml) and IL-1 beta (ED50, 90 pg/ml) were less effective in inhibiting growth. The IL-1-induced growth inhibition was IL-1 receptor mediated, because both IL-1 alpha- and IL-1 beta-mediated growth suppression could be completely reversed by the IL-1 receptor antagonist, the physiological counterpart of IL-1. In the mammosomatotroph GH3 rat pituitary tumor cell line, IL-1 failed to influence growth, indicating that either IL-1 receptors are missing or the IL-1 signaling pathway is uncoupled from the growth regulation. In contrast to growth, in our rat pituitary monolayer cell culture system, IL-1 did not affect ACTH, GH, or PRL secretion as described previously. This discrepancy suggests the involvement of different mechanisms in the IL-1-induced mediation of growth and hormone secretion.