Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

Regulation of hormone secretion in primary cell cultures of human somatotropinomas]

The effects of somatostatin and thyroliberin (thyrotropin-releasing hormone; TRH) on growth hormone (GH) and prolactin (PRL) secretion were studied in short-term (0.5-3h) or long-term (21-24h) incubations using monolayer cell cultures of somatotropin obtained from surgical material of patients with acromegaly. High sensitivity of both GH and PRL release to inhibitory action of somatostatin (10(-11) M) was established. We could not reveal the unambiguous influence of TRH on somatotropic function in the in vivo and in vitro conditions, as compared to the action of this tripeptide on PRL secretion. The results obtained permit us to propose that cell cultures of pituitary adenomata represent adequate and convenient models for studying the pathogenesis of tumor processes in the pituitary gland and for the development of new procedures of pharmacotherapy.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.     

Effects of TRH on cell proliferation of rat pituitary cells (GH3)

Chronic treatment (more than 3 d) of GH3 cells, cloned rat pituitary cells producing prolactin, with 100 nM TRH resulted in a 41% reduction in the rate of cell growth in a medium containing 0.5% fetal bovine serum. These effects of TRH appeared both in the medium containing a higher concentration of serum and in that containing six growth factors, i.e. insulin, transferrin, parathyroid hormone, fibroblast growth factor, triiodothyronine, and multiplication-stimulating activity (MSA) instead of serum. TRH stimulated prolactin production by GH3 cells in a dose-dependent manner both in the serum-supplemented and serum-free media. On the other hand, TRH, at 1 nM, elicited a 130% stimulation in the cellular growth, whereas, at concentrations of more than 10 nM, it inhibited the growth significantly. In the defined culture system, it was demonstrated that TRH stimulated prolactin production in the presence or absence of six growth factors, whereas its inhibitory effects on cellular growth appeared only in the presence of MSA regardless of the presence or absence of the other five factors. Furthermore, it was shown that a dose-dependent stimulatory effect of MSA on the growth of GH3 cells was suppressed by TRH. TRH exhibited only a stimulatory effect on cellular growth in the medium containing the five factors other than MSA. In conclusion, TRH could inhibit cell growth of GH3 in the presence of MSA in the defined medium or MSA-like factor(s) in the serum-supplemented medium.     

Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells.

We examined whether mitogen-activated protein (MAP) kinase is activated by thyrotropin-releasing hormone (TRH) in GH3 cells, and whether MAP kinase activation is involved in secretion of prolactin from these cells. Protein kinase inhibitors--such as PD098059, calphostin C, and genistein--and removal of extracellular Ca2+ inhibited MAP kinase activation by TRH. A cAMP analogue activated MAP kinase in these cells. Effects of cAMP on MAP kinase activation were inhibited by PD098059. TRH-induced prolactin secretion was not inhibited by levels of PD098059 sufficient to i activation but was inhibited by wortmannin (1 microM) and KN93. Treatment of GH3 cells with either TRH or cAMP significantly inhibited DNA synthesis and induced morphological changes. The effects stimulated by TRH were reversed by PD098059 treatment, but the same effects stimulated by cAMP were not. Treatment of GH3 cells with TRH for 48 h significantly increased the prolactin content in GH3 cells and decreased growth hormone content. The increase in prolactin was completely abolished by PD098059, but the decrease in growth hormone was not. These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis and differentiation of GH3 cells, but not in prolactin secretion.     

Growth hormone and prolactin response to thyrotropin releasing hormone and growth hormone releasing factor in the immature turkey

Synthetic thyrotropin releasing hormone (TRH) and human pancreatic growth hormone releasing factor (hpGRF) stimulated growth hormone (GH) secretion in 6- to 9-week-old turkeys in a dose-related manner. TRH and hpGRF (1 and 10 micrograms/kg, respectively) each produced a sixfold increase in circulating GH levels 10 min after iv injection. Neither TRH nor hpGRF caused a substantial change in prolactin (PRL) secretion in unrestrained turkeys sampled through intraatrial cannulas. However, some significant increases in PRL levels, possibly related to stress, were noted.     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

GH4 pituitary cell variants selected as nonresponsive to thyrotropin-releasing hormone-enhanced substratum adhesion are nonresponsive to epidermal growth factor: evidence for a common signaling defect

Thyrotropin-releasing hormone (TRH) and epidermal growth factor both enhance prolactin synthesis and substrate adhesion (a morphological change called stretching) of GH4 rat pituitary cells. We have examined TRH- and EGF-induced cell stretching using genetic and pharmacologic approaches. We selected and isolated a series of GH4 cell variants nonresponsive to TRH-induced cell stretching (str-). This selection yielded several variants that were nonresponsive to both TRH- and EGF-induced stretching but were still responsive to stretching induced by several other agents (tetradecanoylphorbol acetate [TPA], butyrate, and Neplanocin A). One of the str- variants (a14) was examined in detail. TRH, EGF, and TPA each enhanced prolactin synthesis in a14 cells, indicating that the a14 variant contained functional receptor binding sites for all 3 ligands as well as the capacity to generate those intracellular signals required for enhanced prolactin synthesis. Because the str- variants were isolated without selective pressure for EGF-induced stretching and because the possibility of more than one selectable mutation in all the variants is unlikely, we suggest that TRH and EGF share a common mechanism to induce cell stretching. We next examined whether the str- variants had a defect in a signaling pathway or in the biochemical endpoint for TRH- and EGF-induced cell stretching. A pharmacologic approach was utilized to investigate the biochemical basis for induced cell stretching. A synthetic Arg-Gly-Asp-Ser tetrapeptide (RGDS), specific for fibronectin and vitronectin adhesion receptors, inhibited TRH-, EGF-, and TPA-induced GH4 cell stretching and attachment to fibronectin- and vitronectin-coated dishes. These results suggest that the interaction between fibronectin and/or vitronectin and their receptor(s) may be a biochemical endpoint by which several agonists induced stretching of GH4 cells. Because the str- variant has RGDS-specific binding sites for fibronectin and vitronectin and responds to some agents that induce cell stretching via an RGDS receptor, we conclude that the a14 str- variant has a defect in an intracellular signaling pathway, shared by TRH and EGF, which induces cell stretching.     

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.     

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with nonhormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK-, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80-142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10(6) cells/24 h and are currently 100 ng PRL and 90 ng GH/10(6) cells/24 h. Parental GH3 rat cells secreted 720 ng PRL and 660 ng GH/10(6) cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.     

Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture

Addition of thyrotropin releasing hormone (TRH) to the medium of 2 clonal strains of functional rat pituitary cells stimulated the production of prolactin and inhibited growth hormone production. There was no effect on cell growth. Stimulation of prolactin production by TRH was detected within 4 hr, it reached a maximum level (2–5 times control) at 24–48 hr and persisted for at least 20 days in the continued presence of TRH. Stimulation was observed with a concentration of TRH as low as 0.10 ng/ml.     

Thyrotropin-releasing hormone and epidermal growth factor regulate iron-regulatory protein binding in pituitary cells via protein kinase C-dependent and -independent signaling pathways

Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H(2)O(2), nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363-27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.     

Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.