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HYPOPHYSEAL CELLS MODEL SYSTEMS: THE "GH" RAT TUMOR-DERIVED C E L L L I N E S AS A T O O L F O R T H E S T U D Y OF GENE EXPRESSION DANIELLE GOURDJI, JEAN-NOEL LAVERRIERE, EMMANUELLE PASSEGUI~ and JEAN-LUC RICHARD Laboratoire de Neuroendocrinologie Cellulaire et Mol~culaire, CNRS URA 1115 Paris, France
INTRODUCTION Endocrine cells of the anterior pituitary gland are under the control of a complex network of external signals of central and peripheral origin (Gourdji, 1985; Tixier-Vidal and Gourdji, 1981). The complexity arises not only from the impressive number of the regulatory factors but also from their intricate interplay. In addition, many receptors are present on more than one of the six different endocrine cell types and/or trigger more than one intracellular mechanism of action. Besides, paracrine interactions take place, including with non-endocrine cells that are also present in the parenchyma. On the other hand, an increasing number of studies demonstrates that external signals altering a variety of cell functions involve the regulation of specific and/or ubiquitous genes expression. Finally, change in a given gene expression may now be the planned target for a drug. As a consequence, considering this level of regulation becomes necessary when testing substances for medical or veterinary purposes. However, elucidating the mechanisms that govern gene expression of the glandular cells of the pituitary appears particularly difficult. By allowing the control of the neuroendocrine environment, cell cultures partially overcome the complexity of the model and offer a convenient approach for such investigations. Almost all types of cell cultures have been applied successfully to anterior pituitaries. Primary cultures of normal pituitaries and, to a lesser extent, of functional tumors maintain the tissuespecific heterogeneity. Such models are suitable for approaches that respect the cellular level, e.g., in situ hybridization. This technique alone or associated with complementary morphological techniques (immunocytochemistry, reverse hemolytic plaque assay, etc.) is highly relevant for the study of certain aspects of gene expression. Nevertheless, in a pharmacological point of view, such models and such techniques are so far limited by the little amount of cells available and the pitfalls in quantitative analysis. Besides, direct correlations between changes in metabolic alterations, receptor occupancy and changes in gene expression are not achievable. The difficulties arising from the cellular heterogeneity are efficiently overcome by using sorted cells, but this actually enhances the limitation in cell number. Address all correspondences to: Dr. D. Gourdji, Laboratoire de Neuroendocrinologie Cellulaire et Mol6culaire, CNRS URA 1115, Coll~ge de France, 11 place M. Berthelot, 75005 Paris, France. Telephone: (331) 44-27-15-92. Telecopy: (331) 44-27-10-84. Key Words: pituitary cell lines, RNA, Prolactin, Growth Hormone, fos, jun, Secretogranin 1.
Cell Biology and Toxicology, Vol. 8, Copyright © 1992 Princeton Scientific ISSN: 0742-2091
No. 3, pp. 29-38 Publishing Co., Inc.
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An alternative is to use established cell lines that offer homogeneous populations of cells carrying multiple differentiated functions of their normal counterpart, in spite of their frequent tumoral origin (Tashjian, 1979). Assuming that the clonal cell populations are homogeneous, a pharmacological analysis at the level of gene expression by methods that disrupt cell integrity is then convenient. This permits for example the kinetic- and dose-response analysis of multiple RNA species to external agents, provided the specific probes are available. During the last two decades rat "GH" (GH1, GH3, GH3B6, GH4C1, GC) and mouse AtT/20 cell lines have been and are extensively used for studying the neuroendocrine control of PRL, GH and ACTH release by physiological ligands and by drugs, at the cellular and molecular levels. They constitute now invaluable model systems for unraveling the principal mechanisms of endogenous or transfected gene regulation in response to external signals (Gourdji et al., 1982; Tashjian, 1979). As concerns the other major cell types of the anterior pituitary, the models available appear not so practical or fruitful. The TtT mouse thyrotrope cells constitute an excellent model for the study of the TSH gene and its regulation (see in Straub et al., 1990). Nevertheless TtT cells can be maintained in cultured for a limited number of subcultures only, being essentially propagated as ectopic tumors obtained by re-injection of cultured cells in thyroidectomized hosts. Concerning the gonadotrope secreting cells, a cell line has been established from a rat tumor (RC-4B/C) but the LH/FSH secreting cells constitute only a minor percentage of the population, in routine culture conditions (Polkowska et al., 1991). However immortalization of functional gonadotropes by targeted oncogenesis in transgenic mice was recently achieved by P. Mellon, R.Weiner and co-workers illustrating a novel promising strategy. The oncogene used was SV40 large T antigen associated to the 5'flanking promoter of the human glycoprotein hormone alpha-subunit gene to ensure specific targeting of glycoprotein producing pituitary cells. Cells from the pituitary tumors obtained were propagated in culture. Some of them (alpha-T3-1, alpha-T4-2) were established and cloned. They express the alpha subunit common to LH, FSH and TSH and secrete the corresponding protein. Their responsiveness to GnRH unravels that they belong to the gonadotrope lineage (Mellon et al., 1991). In our laboratory, we have taken advantages of "GH" cells to study the multihormonal regulation of the prolactin (PRL) and growth hormone (GH) genes. Together with these genes highly specific of specialized cell types of the pituitary, we have looked for the possible coordinate expression of more ubiquitous genes: 1) the gene encoding for secretogranin I (SgI) (chromogranin B) an acidic tyrosine-sulfated secretory protein, constituent of the matrix of dense-core secretory granules in a number of endocrine and neuroendocrine cells, and suspected to play a role in regulated secretion (see review in Wiedenmann and Huttner 1989); 2) nuclear oncogenes, namely cfos etjun.B, that code for DNA binding proteins presumed to play a role of third messenger at the nuclear level (see reviews in Curran and Franza, 1988; Ransone and Verma, 1990). They were studied as possible mediators of hormones or drugs acting on PRL gene expression or PRL release.
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The results presented were chosen to illustrate: 1) the differential responsiveness of the above mentioned genes in response to external signals known to regulate the secretion of PRL and GH hormones; 2) examples of a pharmacological approach of intracellular mechanisms involved in the regulation of gene expression by external signals (Gourdji et al., 1982; Laverriere et al., 1983; Weisman et al., 1987; Laverriere et al., 1988; Laverriere et al., 1989; Gourdji et al., 1991; Laverriere et al., 1991; Richard et al., 1991). MATERIALS AND METHODS
Cell Culture The majority of the experiments were run using GH3B6 cells, a subclone of the GH3 tumourderived rat pituitary cell line and GC cells (Tashjian Jr., 1979; Gourdji et al., 1982; Gourdji, 1985). GH3B6 cells secrete PRL and GH in a 5-10/1 ratio, GC express only the GH gene. Cells were grown as monolayers in serum-supplemented medium but all experiments were performed in a minimum chemically-defined serum-free medium (SF = Ham's F12 medium containing sodium selenate 3 x 10-8 M, transferrin 5 I.tg/ml) supplemented or not with parathyroid hormone (0.5 ng/m) and penicillin plus streptomycin (5 units, 5p.g/ml) (Laven%re et al., 1988; Weisman et al., 1987). Analysis of Specific RNAs All the RNA studied were qualitatively analysed by Northern blotting (Maniatis et al., 1982) using cDNA inserts (rat PRL cDNA (Cooke et al., 1982), rat GH cDNA (Seeburg et al., 1987), gifted by J. Martial (Liege, Belgium) and human SgI cDNA (Benedum et al., 1987) gifted by W. Huttner (EMBL, Heidelberg) or oligonucleotide probes (ratjun B, mouse c.fos) obtained from Oncogene Science, USA. The corresponding autoradiograms were quantified by computerized densitometry. Sgl, PRL and GH mRNA were also submitted to dot-blotting assays that were quantified by counting the associated radioactivity. In the case of PRL and GH mRNA the concentration could be expressed in absolute values, i.e., megacopies per ~tg total RNA. This was done by including in each blot serial dilutions of GH and PRL mRNAs standards obtained by in vitro transcription using SP6 polymerase (see in Laverriere et al., 1991). Finally, PRL gene transcription rates were also determined by quantifying elongating PRL mRNA, transcribed in isolated nuclei (Laverriere et al., 1988). PRL Hormone and Total Cell Protein Assay Medium PRL was radio-immunoassayed and expressed in nanogram equivalent of the rat PRL RP-3 standard provided by the Hormone Distribution Program of the National Institutes Health. The measurement after 30 minutes reflects the rate of release of stored hormone whereas the measurement after 48h essentially reflects the rate of biosynthesis (cf. Gourdji et al., 1982; Gourdji, 1985). RESULTS
I. Differential Regulation of PRL, GH and SgI mRNA Accumulation by Peripheral Hormones and TRH . A Kinetic and Dose-Dependent Analysis (Laverriere et al., 1991)
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The different regulating factors were chosen on the basis of their long-term effect, i.e., PRL stimulating factors (estradiol-1713,TRH and to a lesser extent the seco-steroid 1,25 dihydroxyvitamin D3 (1,25.D3)) or typical GH stimulating hormones (dexamethasone and T3) (Wark et al., 1983; Gourdji, 1985; Tougard et al., 1988; Lamberts et al., 1990). The results are reported here according to their mode of action: via nuclear receptors for all above mentioned peripheral hormones versus membrane-bound receptors for the neuropeptide.
Time-Dependent Effects of Peripheral Hormones on PRL, GH and SgI mRNA Accumulation (Laverriere et al., 1991) In our experimental conditions, 1713-estradiol (E2)-induced increase PRL mRNA accumulation is rapid and sustained. It is detected within 5 hours and found maximum and stable after 24 hours. In the same cells SgI mRNA is also stimulated as a function of time but with distinct and delayed kinetics: detection after 24 hours and maximal stimulation after 48-72h. On the contrary, GH mRNA accumulation is decreased, with a kinetics corresponding to the stimulation of the PRL gene expression. On the other hand, hormones known to stimulate GH hormone biosynthesis e.g., T3 plus dexamethasone exert opposite effects. As expected they rapidly and drastically stimulate the accumulation of GH mRNA, the maximal stimulation being observed after 24h.They inhibit the accumulation of PRL mRNA even more rapidly, the maximum being reached within 15h. Meanwhile, SgI mRNA levels were also drastically down regulated but with a late timecourse, the inhibition as compared to control increasing continuously as a function of the duration elapsed in serum-free medium.
Dose-dependent Effects of Peripheral Hormones on PRL, GH and Sgl mRNA Accumulation (Laverriere et al., 1991) In spite of their different kinetics, the ED50 of E2 on PRL and SgI mRNA accumulation were found similar (20-60 pM) but the maximal amplitude of stimulation was lower for the SgI gene expression (---xl.33 versus x 1.8). On the contrary, the efficiency and/or the potency of T3 and dexamethasone were found different for each hormone and for each gene. For example: a) dexamethasone was about tenfold more efficient for inhibiting SgI mRNA than stimulating GH mRNA levels (EC 50 : 0.35_+0.16 nM versus 4 + 3 nM); b) dexamethasone (100 nM) alone was able to induce the same maximal down-regulation of Sgl (minus 77 + 3%) than a treatment combining dexamethasone plus T3, whereas the two hormones exerted additive stimulations on GH gene expression; c) dexamethasone alone systematically inhibited PRL mRNA accumulation (minus 71+7 % of control levels) whereas T3 alone elicited either no change or a stimulation. The specificity of the response is further illustrated by the differential responsiveness observed in treatment combining E2 and dexamethasone: in conditions where E2 antagonizes
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dexamethasone-induced PRL mRNA accumulation, the SgI mRNA decrease was prevented only in part and the stimulation of GH mRNA was unchanged. Influence of TRH on PRL and SgI mRNA Accumulation The TRH-induced stimulation of PRL mRNA is now classical (Laverriere et al., 1983; Laverriere et al., 1988; Laverriere et al., 1991; Richard et al., 1991).This essentially originates in a rapid stimulation of the PRL gene transcription. The coupling between TRH receptors to such effect was established in term of dose-dependency (EC 50 --- 0.5-1nM, E max = 10 nM) and kinetics (Laverriere et al., 1983). The kinetic pattern of TRH-induced PRL mRNA accumulation is bell-shaped, being detectable within 4-5 hours, and maximal after 15-20 hours ranging levels 2-4 fold control values, depending on the experiments. Nevertheless, unlike estradiol-1713, TRH regulates the accumulation of PRL mRNA and SgI mRNA in opposite manner in most experiments. This negative regulation is induced with very slow kinetics, i.e., significant after 48h only and modest efficiency (mean decrease = 35%). In the same conditions GH mRNA levels were not significantly altered by TRH (Laverriere et al., 1991; Richard et al., 1991). This contrasts with the TRH-induced decrease previously observed in cells treated in a serum-supplemented culture medium (Laverriere et al., 1983). Interaction of TRH with 1,25.dihydroxyvitamin D3 on PRL and GH Gene Expression (Richard et al., 1991) A delayed but long-lasting stimulating effect of 1,25.D3 on PRL mRNA and PRL secretion was previously reported using "GH" cells. Interestingly, this was observed in a serum-free culture medium only (Wark et al., 1983). This led us to look for a possible interaction of 1,25.D3 on TRH effect on PRL mRNA accumulation as an example of steroid-neuropetide interaction (Richard et al., 1991). We observed that 1,25.D3 potentializes TRH induced PRL gene expression. In conditions where 1,25.D3 alone had no significant effect on PRL mRNA level, it increased TRH (50 nM)-induced PRL mRNA accumulation as well as long-term PRL production in a dosedependent manner. A kinetic study revealed that the effect of 1,25.D3 is delayed, a 48h treatment being required to observe such a positive interaction between the two hormones. Nevertheless, 1,25.D3 does not significantly alter the kinetic pattern of TRH action. These effects are specific of PRL mRNA as compared to GH mRNA, the levels of which did not change significantly, all along the kinetics (Richard et al., 1991; and unpublished). H. Search for Mechanisms of Action Involved in the Regulation of the PRL Gene Expression Search for second messengers involved by TRH action at the PRL gene level (Laverriere et al., 1988) The major initial mechanisms coupling TRH receptors to acute PRL release are almost unraveled (Gershengorn, 1989): activation of a GTP-binding protein, activation of phospholipase C and ensuing catabolism of phosphatidyl-inositol, generation of IP3 that
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mobilizes intracellular calcium stores and diacylglycerol that stimulates protein kinases C, entry of extracellular calcium. This served us as a pilot model to elucidate the mechanisms responsible for TRH-induced PRL gene expression (Laverriere et al., 1983; Laverriere et al., 1988). Actually, using intracellular calcium ionophores and PKC-activating drugs it was possible to demonstrate that both branches of the phosphatidyl inositol cascade likely couple TRH receptors to PRL gene transcription as they mediate the release of stored hormone (Laverriere et al., 1988). These investigations nevertheless revealed some discrepancies between these two parameters. TPA activates PRL gene transcription in a bell-shaped manner versus a linear one for PRL release, and the maximal TPA-induced PRL gene activation is modest (-- 50%) as compared to TRH. On the contrary a preferential role for calcium as mediator of TRH-induced PRL gene transcription was demonstrated using the calcium-channel agonist BAY K 8644. Interestingly, the use of the calcium-channel antagonist PN 200-110 in combination with TRH, TPA or BAY K 8644 suggests that slow voltage-sensitive calcium channels are not necessarily involved in TRH-mediated activation of the PRL gene transcription. Basal PRL gene expression, on the contrary, appears dependent on the activity of these channels that do not significantly regulate the expression of the GH gene (Laverriere et al., 1988).
Search for a possible role of fos/jun proto-oncogenes in the regulation of PRL gene expression and PRL secretion. It is now proposed that a number of cellular (or proto-) oncogenes, namely immediate early genesfos andjun, could play a role in signal transduction from cell surface to the nucleus and thereby participate in multiple cellular responses elicited by hormones, neuronal messages and growth factors (Curran and Franza, 1988; Gutman and Wasylyk, 1991; Ransone and Verma, 1990). Schematically, this means that immediate early genes, typically activated very rapidly, code for regulatory proteins supposed to control and coordinate the expression of subsets of genes, activated or repressed over a frame of hours or days to ensure complex biological responses. This led us to look for possible links betweenfos andjun proto-oncogenes and the neuroendocrine control of GH3B6 cells. In a first step we have demonstrated that TRH was indeed able to augment the accumulation of c.fos RNA in GH3B6 cells, in a dose-dependent manner (Weisman et al., 1987; Weisman et al., 1988). The kinetics of this stimulation is biphasic, with an initial burst occuring within 30 minutes, ranging up to 20 fold above control values, followed by a lower plateau phase sustained for several hours. As compared to the secretory response, c.fos mRNA accumulation occurs within the duration of TRH-induced PRL release, and precedes the activation of the PRL gene. Of note, this was observed in culture conditions where TRH does not promote cell proliferation. Such findings questioned for a possible role of c.fos gene in the stimulation of either the PRL gene or hormone release process. To test these hypotheses, GH3B6 cells were exposed to peptides or drugs known to regulate PRL gene expression or PRL release through different mechanisms.
Hypophyseal Cells Model Systems 35
The results indicate that, the phorbol ester TPA, the calcium ionophore ionomycin and the calcium channel agonist Bay K8644 were all able to increase c.fos mRNA levels in a direct dose-dependent manner to the following maximal levels: 2-3-fold above control for the calcium activating drugs (lktM) and 6-10-fold above control for TPA (1.6~tM). These stimulations suggest that similarly to the PRL gene and PRL release, the level of c.fos gene expression is dependent on the Ca2+ and protein kinase C (PKC)-dependent events triggered by TRH in GH3/B6 cells. The dose-effect of TPA and treatments combining TPA or TRH plus drugs activating calcium-dependent pathways further supported the parallelism between the regulation of c.fos mRNA levels and PRL release (Gourdji et al., 1988). Some experimental conditions that revealed that PRL depletion is not a signal sufficient to stimulate PRL gene expression also unraveled discrepancies in the control of the c.fos gene and PRL gene expression: 1- KCI 30 mM stimulates both PRL release and c.fos mRNA levels without altering PRL mRNA levels, 2- VIP and somatostatin modulate the accumulation of c.fos mRNA in parallel with their opposite effects on PRL release while they do not modify PRL synthesis (Gourdji et al., 1990; Gourdji et al., 1991). On the other hand it is clear from literature that c.fos gene product cannot bind DNA and thus regulate transcription unless dimerised with a member of the JUN family. The resulting FOS/JUN heterodimers (AP1 complexes) constitute specific and efficient transcriptional factors (see Curran and Franza, 1988; Ransone and Verma, 1990). The screening of RNA samples disclosed a differential expression of the diverse members of thejun family in GH3B6 cells that led us to primarily focus our investigations on jun B (Passegu6 et al., submitted). The results establish that similarly to c.fos, jun B mRNA accumulation is strongly stimulated by TRH in a dose- and time-dependent manner. The induced stimulation is very rapid (10 minutes) and present a biphasic pattern. As compared to c.fos changes, the peak culminates at lower levels and is slightly delayed while the plateau phase is similarly detectable up to 24h at least. The effects of ionomycin, KC1 and TPA were also analyzed. They indicate that calciumdependent, protein kinase C-and PKA -dependent mechanisms are susceptible to strongly activate jun B mRNA accumulation and in a manner similar to hormone release (Gourdji et al., 1991; and Passegu6 et al., submitted). Altogether, these studies reveal the coordinated regulation of c.fos and jun B genes expression in response to external signals that control GH3B6 secretion. Such a finding questions a possible role of third messenger for the heterodimer c.FOS/JUN B in the neuroendocrine control of these ceils. DISCUSSION AND CONCLUSIONS The results summarized here illustrate some pharmacological characteristics of the regulation of a small number of genes expressed and regulated in functional pituitary tumor cell lines, in a manner coordinated with the regulation of the secretory activity. Of note, the specificity of
36 Gourdji et al.
each gene resides not only in qualitative and quantitative responsiveness to a given external signal but also in the time-course of the changes observed. For example these studies illustrate the differential regulation of the PRL and GH genes by steroid and thyroid hormones whereas the SgI gene expression appears controlled almost in parallel with the PRL gene, although in an attenuated and delayed manner. The latter characteristics contrast with the drastic, acute initial changes observed at the level of c.fos and jun B mRNA levels elicited by secondmessengers-dependent mechanisms. This infers: 1) that the analysis of pharmacologicallyinduced changes of gene expression is presently achievable using such or similar models; 2) that the changes might be detectable within minutes or within days and can be transient or sustained. Search for investigating the regulation of resident proteins by external signal was here restricted to the investigation of ubiquitous transcriptional factors such asfos/jun oncogenes. It demonstrates temporal and pharmacological relations but no causal link is indicated yet. Nevertheless such investigations can be extended to the regulation of tissue specific transactivating factors such as Pit-1/GH-F1 (Ingram et al., 1990; He and Rosenfeld, 1991; Frawley and Boockfor, 1991; Yan et al., 1991). The cross-talk now assumed to occur at the level of transduction mechanisms possess their counterpart at the level of gene regulation: existence of numerous transcription factors, ability of some DNA-responsive element to interact with several transactivating factors, interaction between transactivating factors of the same family (e.g., fos/jun), and between members of different family (e.g., AP1/steroid or thyroid hormone receptors) (He and Rosenfeld, 1991). Thus pharmacological investigation to the mode of action at the gene level cannot be limited to the simple measurement of endogenous genes as above described. The obvious complementary approach consists in the study of transfected artificially modified genes. "GH" pituitary tumor cell lines constitute here also an excellent model. Not only do they possess the functional receptors for external signals, but they express tissue specific transcriptional factors such as Pit-1/GH-F1 necessary to the expression and optimal regulation of genes like PRL or GH genes. In this context, they offer advantageous model systems for investigating the regulations specific to the human GH and PRL genes (Morin et al., 1990; Peers et al., 1990) and thus could be considered for pharmacological screenings of drugs. ACKNOWLEDGMENTS These works were supported by the CNRS (UA1115) and grants from ARC (# 600084) and INSERM (CL # 874005). REFERENCES
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COOKE, N.E., COIT, D., WEINER, R.I., BAXTER, J.D., and MARTIAL, J.A. (1982). Structure of cloned DNA complementary to rat prolactin messenger RNA. J. BIOL. CHEM. 255:6502-6506. CURRAN, T. and FRANZA, R. (1988). Fos and Jun: the AP1 connection. CELL. 55:395-397. FRAWLEY, L.S. and BOOCKFOR, F.R. (1991). Mammosomatotropes: presence and functions in normal and neoplastic tissue. ENDOCR. REV. 12:337-355. GERSHENGORN, M.C. (1989). "Role of inositol lipid lecond messengers in regulation of secretion: studies of Thyrotropin-releasing Hormone action in pituitary cells." In: Secretion and its control, pp. 1-15, The Rockefeller University Press, New York. GOURDJI, D. (1985). Multihormonal regulation of the pituitary gland: binding and secretory responses to hypothalamic neuropeptides in rat GH pituitary strains in culture. NEUROCHEM. INT. 7:979-994. GOURDJI, D., BUISSON, N., WEISMAN, A.S., and LAVERRIERE, J.N. (1990). A. Tixier-Vidal c.fos proto-oncogene expression and prolactin release share common neuroendocrine control. Neuroendocrinology, 2nd International Congress of Neuroendocrinology, Bordeaux France, June 24-29, Abstract, S 15.3, p34. GOURDJI, D., LAVERRIERE, J.N., PASSEGUE, E. BUISSON, N., and RICHARD, J.L. (1991). R~gulations multig~niques et activit~s s~cr~toires dans des cellules mammosomatotropes. XX~me Congr~s de la Soci6t6 de Neuroendocrinologie Experimentale. Ann. Endocrinol. 52:11N. GOURDJI, D., LAVERRIERE, J.N., WEISMAN, A.S., BUISSON, N., and TIXIER-VIDAL, A. (1989). c.fos mRNA and PRL release are regulated in parallel in GH3B6 rat pituitary tumor cells. 71st Annual Meeting of the Endocrine Society, 1989, 827. GOURDJI, D., LAVERRIERE, J.N., WEISMAN, A.S., BUISSON, N., and TIXIER-VIDAL, A. (1988). "Coordinate expression of genes in relation with prolactin secretion." In: Prolactin gene family and its receptors. Molecular biology to clinical problems (K. Hoshino, ed.), pp. 227235, Elsevier Science Publishers, Amsterdam. GOURDJI, D., TOUGARD, C., and TIXIER-VIDAL, A. (1982). "Clonal prolactin strains as a tool in neuroendocrinology." In: Frontiers in Neuroendocrinology, (W.F. Ganong and L. Martini, eds., Raven Press), 7, pp. 317-357, New York. GUTMAN, A. and WASYLYK, B. (1991). Nuclear targets for transcription regulation by oncogenes. TIG vol 7, 2:49-54. INGRAHAM, H.A., ALBERT, V.R., CHEN, R., CRENSHAW III, B., ELSHOLTZ, H.P., HE, X., KAPILOFF, M.S., MANGALAM, H.J., SWANSON, L.W., TREACY, M.N., and ROSENFELD, M.G. (1990). A family of POU-domain and pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development. Annu. Rev. Physiol. 52:773-791. HE, X. and ROSENFELD, M.G. (1991). Mechanisms of complex transcriptional regulation: implication for brain development. Neuron. 7:183-196. LAMBERTS, S.W.J. and MCLEOD, M. (1990). Regulation of prolactin secretion at the level of the lactotroph. Physiol. Rev. 70:279-318. LAVERRIERE, J.N., MORN, A., TIXIER-V/DAL, A., TRUONG, A.T., GOURDJI, D., and MARTIAL, J.A. (1983). Inverse control of prolactin and growth hormone gene expression: effect of thyroliberin on transcription and RNA stabilization. EMBO J. 2:1493-1499. LAVERRIERE, J.N., RICHARD, J.L., BUISSON, N.B., MARTIAL, J.A., TIXIER-VIDAL, A., and GOURDJI, D. (1989). Thyroliberin and dihydropyridines modulate prolactin gene expression through interacting pathways in GH3 cells. Neuroendocrinology. 50:693-701. LAVERRIERE, J.N., RICHARD, J.L., MORIN, A., BUISSON, N.B., TIXIER-VIDAL, A., HUTTNER, W., and GOURDJI, D. (1991). Secretogranin I (chromogranin B) mRNA accumulation is hormonally regulated in GH3B6 rat pituitary tumor cells. Mol. Cel. Endocr. 80:41-51. LAVERRIERE, J.N., TIXIER-VIDAL, A., BUISSON, N.B., MORIN, A., MARTIAL, J.A., and GOURDJI, D. (1988). Preferential role of calcium in the regulation of prolactin gene transcription by thyrotropin-releasing hormone in GH3 pituitary cells. Endocrinolgy. 122:333-340. MANIATIS T., FRITSCH, E.F., and SAMBROOK, J. (1982). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
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MELLON, P.L., WINDLE, J.J., WEINER, R.I. (1991). Immortalization of neuroendocrine cells by targeted oncogenesis. Rec. Prog. Horm. Res. 47:69-96. MORIN, A., LOUETTE, J., VOZ, M.L.J., TIXIER-VIDAL, A., BELAYEW, A., and MARTIAL, J.A. (1990). Triiodothyronine inhibits transcription from the human growth hormone promoter. Mol. Cel. Endocr. 71:261-267. PEERS, B., VOZ, M.L., MONGET P., MATHY-HARTERT, M., BERWAER, M., BELAYEW, A., and MARTIAL, J.A. (1990). Regulatory elements controlling pituitary-specific expression of the human prolactin gene. Mol. Cell. Biol. 10:4690-4700. POLKOWSKA, J., BERAULT, A., HURBAIN-KOSMATH, I., JOLLY, G., and JUTISZ, M. (1991). Bihormonal producing gonadotropins and prolactin in a rat pituitary cell line (RC-4B/C). Neuroendocrinology. 54:263-73. RANSONE, L.J. and VERMA, I.M. (1990). Nuclear proto-oncogenes fos and jun. Annu. Rev. Cell Biol. 539-573. RICHARD, J.L., LAVERRIERE, J.N., BUISSON, N., TIXIER-VIDAL, A., and GOURDJI, D. (1991). "Kinetics of TRH and 1,25 dihydroxyvitamin D3 (1,25.D3) interaction on the expression of PRL and GH genes in GH3B6 pituitary ceils." In: Vitamin D: gene regulation, structure function analysis, and clinical application; Proceedings of the Eight Workshop on vitamin D (Paris). (A.W. Norman, R. Bouillon, M. Thomasset, eds.) pp. 28-30, Waiter de Gruyter, Berlin. SEEBURG, P.H., SHINE, J.A., BAXTER, J.D., and GOODMAN, H.M. (1987). Nucleotide Sequence and amplification in bacteria of structural gene for rat growth hormone. Nature. 270:486-488. STRAUB, R.E., FRECH, G.C., JOHO, R,H., and GERSHENGORN, M.C. (1990). Expression cloning of a eDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc. Natl. Acad. Sci. USA. 87:9514-9518 TASHAIAN JR., A.H. (1979). Clonal strains of hormone producing pituitary cells . In: Methods In Enzymology (W.B. Jacoby, I.H. Pastan, eds.) vol 58, pp. 527-535. Academic Press, New York. TIXIER-VIDAL, A. and GOURDJI, D. (1981). Mechanism of action of synthetic hypothalamic peptides on anterior pituitary cells. Physiol. Rev. 61:974-1011 TOUGARD, C. and TIXIER-VIDAL, A. (1988). Lactotropes and gonadotropes. In: The Physiology of Reproduction (E. Knobil and J. Neill, eds.), pp. 1305-1333. Raven Press, NewYork. WARK, J.D. and TASHJIAN, A.H. (1983). Regulation of Prolaetin mRNA by 1,25Dihydroxyvitamin D3 in GH4.CI Cells. J. B. Chem. 258:12118-12121. WEISMAN, A.S., TIXIER-VIDAL, A., and GOURDJI, D. (1987). Thyrotropin-releasing hormone increases the levels of c-Fos and beta-actin mRNA in GH3B6 pituitary tumor cells. In: Vitro Cellular and Developmental Biology. 23:585-590 WEISMAN, A.S., TIXIER-VIDAL, A., and GOURDJI, D. (1988)."c-fos and actin gene expression is regulated by thyrotropin releasing hormone and serum in GH3/B6 pituitary tumor cells." In: Progress in Cancer Research and Theraphy, (F. Bresciani, R.J.B. King, M.E. Lippman, and J.P. Raynaud, eds.) vol. 35: Hormone and Cancer 3, Raven Press, LTD., New York. WIEDENMANN, B. and HUTTNER, W.B. (1989). Synaptophysin and Chromagranins/ Secretogranins-widespread constituents of distinct types of neuroendocrine vesicles and new tolls in tumor diagnosis. Virchows Archiv B Cell Pathol. 58:95-121. YAN, G., PAN, W.T., and BANCROFT, C. (1991). Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein PIT-1. Molecular Endocrinology. 5:535541.
HYPOPHYSEAL CELLS MODEL SYSTEMS: THE "GH" RAT TUMOR-DERIVED C E L L L I N E S AS A T O O L F O R T H E S T U D Y OF GENE EXPRESSION DANIELLE GOURDJI, JEAN-NOEL LAVERRIERE, EMMANUELLE PASSEGUI~ and JEAN-LUC RICHARD Laboratoire de Neuroendocrinologie Cellulaire et Mol~culaire, CNRS URA 1115 Paris, France
INTRODUCTION Endocrine cells of the anterior pituitary gland are under the control of a complex network of external signals of central and peripheral origin (Gourdji, 1985; Tixier-Vidal and Gourdji, 1981). The complexity arises not only from the impressive number of the regulatory factors but also from their intricate interplay. In addition, many receptors are present on more than one of the six different endocrine cell types and/or trigger more than one intracellular mechanism of action. Besides, paracrine interactions take place, including with non-endocrine cells that are also present in the parenchyma. On the other hand, an increasing number of studies demonstrates that external signals altering a variety of cell functions involve the regulation of specific and/or ubiquitous genes expression. Finally, change in a given gene expression may now be the planned target for a drug. As a consequence, considering this level of regulation becomes necessary when testing substances for medical or veterinary purposes. However, elucidating the mechanisms that govern gene expression of the glandular cells of the pituitary appears particularly difficult. By allowing the control of the neuroendocrine environment, cell cultures partially overcome the complexity of the model and offer a convenient approach for such investigations. Almost all types of cell cultures have been applied successfully to anterior pituitaries. Primary cultures of normal pituitaries and, to a lesser extent, of functional tumors maintain the tissuespecific heterogeneity. Such models are suitable for approaches that respect the cellular level, e.g., in situ hybridization. This technique alone or associated with complementary morphological techniques (immunocytochemistry, reverse hemolytic plaque assay, etc.) is highly relevant for the study of certain aspects of gene expression. Nevertheless, in a pharmacological point of view, such models and such techniques are so far limited by the little amount of cells available and the pitfalls in quantitative analysis. Besides, direct correlations between changes in metabolic alterations, receptor occupancy and changes in gene expression are not achievable. The difficulties arising from the cellular heterogeneity are efficiently overcome by using sorted cells, but this actually enhances the limitation in cell number. Address all correspondences to: Dr. D. Gourdji, Laboratoire de Neuroendocrinologie Cellulaire et Mol6culaire, CNRS URA 1115, Coll~ge de France, 11 place M. Berthelot, 75005 Paris, France. Telephone: (331) 44-27-15-92. Telecopy: (331) 44-27-10-84. Key Words: pituitary cell lines, RNA, Prolactin, Growth Hormone, fos, jun, Secretogranin 1.
Cell Biology and Toxicology, Vol. 8, Copyright © 1992 Princeton Scientific ISSN: 0742-2091
No. 3, pp. 29-38 Publishing Co., Inc.
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An alternative is to use established cell lines that offer homogeneous populations of cells carrying multiple differentiated functions of their normal counterpart, in spite of their frequent tumoral origin (Tashjian, 1979). Assuming that the clonal cell populations are homogeneous, a pharmacological analysis at the level of gene expression by methods that disrupt cell integrity is then convenient. This permits for example the kinetic- and dose-response analysis of multiple RNA species to external agents, provided the specific probes are available. During the last two decades rat "GH" (GH1, GH3, GH3B6, GH4C1, GC) and mouse AtT/20 cell lines have been and are extensively used for studying the neuroendocrine control of PRL, GH and ACTH release by physiological ligands and by drugs, at the cellular and molecular levels. They constitute now invaluable model systems for unraveling the principal mechanisms of endogenous or transfected gene regulation in response to external signals (Gourdji et al., 1982; Tashjian, 1979). As concerns the other major cell types of the anterior pituitary, the models available appear not so practical or fruitful. The TtT mouse thyrotrope cells constitute an excellent model for the study of the TSH gene and its regulation (see in Straub et al., 1990). Nevertheless TtT cells can be maintained in cultured for a limited number of subcultures only, being essentially propagated as ectopic tumors obtained by re-injection of cultured cells in thyroidectomized hosts. Concerning the gonadotrope secreting cells, a cell line has been established from a rat tumor (RC-4B/C) but the LH/FSH secreting cells constitute only a minor percentage of the population, in routine culture conditions (Polkowska et al., 1991). However immortalization of functional gonadotropes by targeted oncogenesis in transgenic mice was recently achieved by P. Mellon, R.Weiner and co-workers illustrating a novel promising strategy. The oncogene used was SV40 large T antigen associated to the 5'flanking promoter of the human glycoprotein hormone alpha-subunit gene to ensure specific targeting of glycoprotein producing pituitary cells. Cells from the pituitary tumors obtained were propagated in culture. Some of them (alpha-T3-1, alpha-T4-2) were established and cloned. They express the alpha subunit common to LH, FSH and TSH and secrete the corresponding protein. Their responsiveness to GnRH unravels that they belong to the gonadotrope lineage (Mellon et al., 1991). In our laboratory, we have taken advantages of "GH" cells to study the multihormonal regulation of the prolactin (PRL) and growth hormone (GH) genes. Together with these genes highly specific of specialized cell types of the pituitary, we have looked for the possible coordinate expression of more ubiquitous genes: 1) the gene encoding for secretogranin I (SgI) (chromogranin B) an acidic tyrosine-sulfated secretory protein, constituent of the matrix of dense-core secretory granules in a number of endocrine and neuroendocrine cells, and suspected to play a role in regulated secretion (see review in Wiedenmann and Huttner 1989); 2) nuclear oncogenes, namely cfos etjun.B, that code for DNA binding proteins presumed to play a role of third messenger at the nuclear level (see reviews in Curran and Franza, 1988; Ransone and Verma, 1990). They were studied as possible mediators of hormones or drugs acting on PRL gene expression or PRL release.
Hypophyseal Cells Model Systems 31
The results presented were chosen to illustrate: 1) the differential responsiveness of the above mentioned genes in response to external signals known to regulate the secretion of PRL and GH hormones; 2) examples of a pharmacological approach of intracellular mechanisms involved in the regulation of gene expression by external signals (Gourdji et al., 1982; Laverriere et al., 1983; Weisman et al., 1987; Laverriere et al., 1988; Laverriere et al., 1989; Gourdji et al., 1991; Laverriere et al., 1991; Richard et al., 1991). MATERIALS AND METHODS
Cell Culture The majority of the experiments were run using GH3B6 cells, a subclone of the GH3 tumourderived rat pituitary cell line and GC cells (Tashjian Jr., 1979; Gourdji et al., 1982; Gourdji, 1985). GH3B6 cells secrete PRL and GH in a 5-10/1 ratio, GC express only the GH gene. Cells were grown as monolayers in serum-supplemented medium but all experiments were performed in a minimum chemically-defined serum-free medium (SF = Ham's F12 medium containing sodium selenate 3 x 10-8 M, transferrin 5 I.tg/ml) supplemented or not with parathyroid hormone (0.5 ng/m) and penicillin plus streptomycin (5 units, 5p.g/ml) (Laven%re et al., 1988; Weisman et al., 1987). Analysis of Specific RNAs All the RNA studied were qualitatively analysed by Northern blotting (Maniatis et al., 1982) using cDNA inserts (rat PRL cDNA (Cooke et al., 1982), rat GH cDNA (Seeburg et al., 1987), gifted by J. Martial (Liege, Belgium) and human SgI cDNA (Benedum et al., 1987) gifted by W. Huttner (EMBL, Heidelberg) or oligonucleotide probes (ratjun B, mouse c.fos) obtained from Oncogene Science, USA. The corresponding autoradiograms were quantified by computerized densitometry. Sgl, PRL and GH mRNA were also submitted to dot-blotting assays that were quantified by counting the associated radioactivity. In the case of PRL and GH mRNA the concentration could be expressed in absolute values, i.e., megacopies per ~tg total RNA. This was done by including in each blot serial dilutions of GH and PRL mRNAs standards obtained by in vitro transcription using SP6 polymerase (see in Laverriere et al., 1991). Finally, PRL gene transcription rates were also determined by quantifying elongating PRL mRNA, transcribed in isolated nuclei (Laverriere et al., 1988). PRL Hormone and Total Cell Protein Assay Medium PRL was radio-immunoassayed and expressed in nanogram equivalent of the rat PRL RP-3 standard provided by the Hormone Distribution Program of the National Institutes Health. The measurement after 30 minutes reflects the rate of release of stored hormone whereas the measurement after 48h essentially reflects the rate of biosynthesis (cf. Gourdji et al., 1982; Gourdji, 1985). RESULTS
I. Differential Regulation of PRL, GH and SgI mRNA Accumulation by Peripheral Hormones and TRH . A Kinetic and Dose-Dependent Analysis (Laverriere et al., 1991)
32 Gourdji et al.
The different regulating factors were chosen on the basis of their long-term effect, i.e., PRL stimulating factors (estradiol-1713,TRH and to a lesser extent the seco-steroid 1,25 dihydroxyvitamin D3 (1,25.D3)) or typical GH stimulating hormones (dexamethasone and T3) (Wark et al., 1983; Gourdji, 1985; Tougard et al., 1988; Lamberts et al., 1990). The results are reported here according to their mode of action: via nuclear receptors for all above mentioned peripheral hormones versus membrane-bound receptors for the neuropeptide.
Time-Dependent Effects of Peripheral Hormones on PRL, GH and SgI mRNA Accumulation (Laverriere et al., 1991) In our experimental conditions, 1713-estradiol (E2)-induced increase PRL mRNA accumulation is rapid and sustained. It is detected within 5 hours and found maximum and stable after 24 hours. In the same cells SgI mRNA is also stimulated as a function of time but with distinct and delayed kinetics: detection after 24 hours and maximal stimulation after 48-72h. On the contrary, GH mRNA accumulation is decreased, with a kinetics corresponding to the stimulation of the PRL gene expression. On the other hand, hormones known to stimulate GH hormone biosynthesis e.g., T3 plus dexamethasone exert opposite effects. As expected they rapidly and drastically stimulate the accumulation of GH mRNA, the maximal stimulation being observed after 24h.They inhibit the accumulation of PRL mRNA even more rapidly, the maximum being reached within 15h. Meanwhile, SgI mRNA levels were also drastically down regulated but with a late timecourse, the inhibition as compared to control increasing continuously as a function of the duration elapsed in serum-free medium.
Dose-dependent Effects of Peripheral Hormones on PRL, GH and Sgl mRNA Accumulation (Laverriere et al., 1991) In spite of their different kinetics, the ED50 of E2 on PRL and SgI mRNA accumulation were found similar (20-60 pM) but the maximal amplitude of stimulation was lower for the SgI gene expression (---xl.33 versus x 1.8). On the contrary, the efficiency and/or the potency of T3 and dexamethasone were found different for each hormone and for each gene. For example: a) dexamethasone was about tenfold more efficient for inhibiting SgI mRNA than stimulating GH mRNA levels (EC 50 : 0.35_+0.16 nM versus 4 + 3 nM); b) dexamethasone (100 nM) alone was able to induce the same maximal down-regulation of Sgl (minus 77 + 3%) than a treatment combining dexamethasone plus T3, whereas the two hormones exerted additive stimulations on GH gene expression; c) dexamethasone alone systematically inhibited PRL mRNA accumulation (minus 71+7 % of control levels) whereas T3 alone elicited either no change or a stimulation. The specificity of the response is further illustrated by the differential responsiveness observed in treatment combining E2 and dexamethasone: in conditions where E2 antagonizes
Hypophyseal Cells Model Systems 33
dexamethasone-induced PRL mRNA accumulation, the SgI mRNA decrease was prevented only in part and the stimulation of GH mRNA was unchanged. Influence of TRH on PRL and SgI mRNA Accumulation The TRH-induced stimulation of PRL mRNA is now classical (Laverriere et al., 1983; Laverriere et al., 1988; Laverriere et al., 1991; Richard et al., 1991).This essentially originates in a rapid stimulation of the PRL gene transcription. The coupling between TRH receptors to such effect was established in term of dose-dependency (EC 50 --- 0.5-1nM, E max = 10 nM) and kinetics (Laverriere et al., 1983). The kinetic pattern of TRH-induced PRL mRNA accumulation is bell-shaped, being detectable within 4-5 hours, and maximal after 15-20 hours ranging levels 2-4 fold control values, depending on the experiments. Nevertheless, unlike estradiol-1713, TRH regulates the accumulation of PRL mRNA and SgI mRNA in opposite manner in most experiments. This negative regulation is induced with very slow kinetics, i.e., significant after 48h only and modest efficiency (mean decrease = 35%). In the same conditions GH mRNA levels were not significantly altered by TRH (Laverriere et al., 1991; Richard et al., 1991). This contrasts with the TRH-induced decrease previously observed in cells treated in a serum-supplemented culture medium (Laverriere et al., 1983). Interaction of TRH with 1,25.dihydroxyvitamin D3 on PRL and GH Gene Expression (Richard et al., 1991) A delayed but long-lasting stimulating effect of 1,25.D3 on PRL mRNA and PRL secretion was previously reported using "GH" cells. Interestingly, this was observed in a serum-free culture medium only (Wark et al., 1983). This led us to look for a possible interaction of 1,25.D3 on TRH effect on PRL mRNA accumulation as an example of steroid-neuropetide interaction (Richard et al., 1991). We observed that 1,25.D3 potentializes TRH induced PRL gene expression. In conditions where 1,25.D3 alone had no significant effect on PRL mRNA level, it increased TRH (50 nM)-induced PRL mRNA accumulation as well as long-term PRL production in a dosedependent manner. A kinetic study revealed that the effect of 1,25.D3 is delayed, a 48h treatment being required to observe such a positive interaction between the two hormones. Nevertheless, 1,25.D3 does not significantly alter the kinetic pattern of TRH action. These effects are specific of PRL mRNA as compared to GH mRNA, the levels of which did not change significantly, all along the kinetics (Richard et al., 1991; and unpublished). H. Search for Mechanisms of Action Involved in the Regulation of the PRL Gene Expression Search for second messengers involved by TRH action at the PRL gene level (Laverriere et al., 1988) The major initial mechanisms coupling TRH receptors to acute PRL release are almost unraveled (Gershengorn, 1989): activation of a GTP-binding protein, activation of phospholipase C and ensuing catabolism of phosphatidyl-inositol, generation of IP3 that
34 Gourdji et al.
mobilizes intracellular calcium stores and diacylglycerol that stimulates protein kinases C, entry of extracellular calcium. This served us as a pilot model to elucidate the mechanisms responsible for TRH-induced PRL gene expression (Laverriere et al., 1983; Laverriere et al., 1988). Actually, using intracellular calcium ionophores and PKC-activating drugs it was possible to demonstrate that both branches of the phosphatidyl inositol cascade likely couple TRH receptors to PRL gene transcription as they mediate the release of stored hormone (Laverriere et al., 1988). These investigations nevertheless revealed some discrepancies between these two parameters. TPA activates PRL gene transcription in a bell-shaped manner versus a linear one for PRL release, and the maximal TPA-induced PRL gene activation is modest (-- 50%) as compared to TRH. On the contrary a preferential role for calcium as mediator of TRH-induced PRL gene transcription was demonstrated using the calcium-channel agonist BAY K 8644. Interestingly, the use of the calcium-channel antagonist PN 200-110 in combination with TRH, TPA or BAY K 8644 suggests that slow voltage-sensitive calcium channels are not necessarily involved in TRH-mediated activation of the PRL gene transcription. Basal PRL gene expression, on the contrary, appears dependent on the activity of these channels that do not significantly regulate the expression of the GH gene (Laverriere et al., 1988).
Search for a possible role of fos/jun proto-oncogenes in the regulation of PRL gene expression and PRL secretion. It is now proposed that a number of cellular (or proto-) oncogenes, namely immediate early genesfos andjun, could play a role in signal transduction from cell surface to the nucleus and thereby participate in multiple cellular responses elicited by hormones, neuronal messages and growth factors (Curran and Franza, 1988; Gutman and Wasylyk, 1991; Ransone and Verma, 1990). Schematically, this means that immediate early genes, typically activated very rapidly, code for regulatory proteins supposed to control and coordinate the expression of subsets of genes, activated or repressed over a frame of hours or days to ensure complex biological responses. This led us to look for possible links betweenfos andjun proto-oncogenes and the neuroendocrine control of GH3B6 cells. In a first step we have demonstrated that TRH was indeed able to augment the accumulation of c.fos RNA in GH3B6 cells, in a dose-dependent manner (Weisman et al., 1987; Weisman et al., 1988). The kinetics of this stimulation is biphasic, with an initial burst occuring within 30 minutes, ranging up to 20 fold above control values, followed by a lower plateau phase sustained for several hours. As compared to the secretory response, c.fos mRNA accumulation occurs within the duration of TRH-induced PRL release, and precedes the activation of the PRL gene. Of note, this was observed in culture conditions where TRH does not promote cell proliferation. Such findings questioned for a possible role of c.fos gene in the stimulation of either the PRL gene or hormone release process. To test these hypotheses, GH3B6 cells were exposed to peptides or drugs known to regulate PRL gene expression or PRL release through different mechanisms.
Hypophyseal Cells Model Systems 35
The results indicate that, the phorbol ester TPA, the calcium ionophore ionomycin and the calcium channel agonist Bay K8644 were all able to increase c.fos mRNA levels in a direct dose-dependent manner to the following maximal levels: 2-3-fold above control for the calcium activating drugs (lktM) and 6-10-fold above control for TPA (1.6~tM). These stimulations suggest that similarly to the PRL gene and PRL release, the level of c.fos gene expression is dependent on the Ca2+ and protein kinase C (PKC)-dependent events triggered by TRH in GH3/B6 cells. The dose-effect of TPA and treatments combining TPA or TRH plus drugs activating calcium-dependent pathways further supported the parallelism between the regulation of c.fos mRNA levels and PRL release (Gourdji et al., 1988). Some experimental conditions that revealed that PRL depletion is not a signal sufficient to stimulate PRL gene expression also unraveled discrepancies in the control of the c.fos gene and PRL gene expression: 1- KCI 30 mM stimulates both PRL release and c.fos mRNA levels without altering PRL mRNA levels, 2- VIP and somatostatin modulate the accumulation of c.fos mRNA in parallel with their opposite effects on PRL release while they do not modify PRL synthesis (Gourdji et al., 1990; Gourdji et al., 1991). On the other hand it is clear from literature that c.fos gene product cannot bind DNA and thus regulate transcription unless dimerised with a member of the JUN family. The resulting FOS/JUN heterodimers (AP1 complexes) constitute specific and efficient transcriptional factors (see Curran and Franza, 1988; Ransone and Verma, 1990). The screening of RNA samples disclosed a differential expression of the diverse members of thejun family in GH3B6 cells that led us to primarily focus our investigations on jun B (Passegu6 et al., submitted). The results establish that similarly to c.fos, jun B mRNA accumulation is strongly stimulated by TRH in a dose- and time-dependent manner. The induced stimulation is very rapid (10 minutes) and present a biphasic pattern. As compared to c.fos changes, the peak culminates at lower levels and is slightly delayed while the plateau phase is similarly detectable up to 24h at least. The effects of ionomycin, KC1 and TPA were also analyzed. They indicate that calciumdependent, protein kinase C-and PKA -dependent mechanisms are susceptible to strongly activate jun B mRNA accumulation and in a manner similar to hormone release (Gourdji et al., 1991; and Passegu6 et al., submitted). Altogether, these studies reveal the coordinated regulation of c.fos and jun B genes expression in response to external signals that control GH3B6 secretion. Such a finding questions a possible role of third messenger for the heterodimer c.FOS/JUN B in the neuroendocrine control of these ceils. DISCUSSION AND CONCLUSIONS The results summarized here illustrate some pharmacological characteristics of the regulation of a small number of genes expressed and regulated in functional pituitary tumor cell lines, in a manner coordinated with the regulation of the secretory activity. Of note, the specificity of
36 Gourdji et al.
each gene resides not only in qualitative and quantitative responsiveness to a given external signal but also in the time-course of the changes observed. For example these studies illustrate the differential regulation of the PRL and GH genes by steroid and thyroid hormones whereas the SgI gene expression appears controlled almost in parallel with the PRL gene, although in an attenuated and delayed manner. The latter characteristics contrast with the drastic, acute initial changes observed at the level of c.fos and jun B mRNA levels elicited by secondmessengers-dependent mechanisms. This infers: 1) that the analysis of pharmacologicallyinduced changes of gene expression is presently achievable using such or similar models; 2) that the changes might be detectable within minutes or within days and can be transient or sustained. Search for investigating the regulation of resident proteins by external signal was here restricted to the investigation of ubiquitous transcriptional factors such asfos/jun oncogenes. It demonstrates temporal and pharmacological relations but no causal link is indicated yet. Nevertheless such investigations can be extended to the regulation of tissue specific transactivating factors such as Pit-1/GH-F1 (Ingram et al., 1990; He and Rosenfeld, 1991; Frawley and Boockfor, 1991; Yan et al., 1991). The cross-talk now assumed to occur at the level of transduction mechanisms possess their counterpart at the level of gene regulation: existence of numerous transcription factors, ability of some DNA-responsive element to interact with several transactivating factors, interaction between transactivating factors of the same family (e.g., fos/jun), and between members of different family (e.g., AP1/steroid or thyroid hormone receptors) (He and Rosenfeld, 1991). Thus pharmacological investigation to the mode of action at the gene level cannot be limited to the simple measurement of endogenous genes as above described. The obvious complementary approach consists in the study of transfected artificially modified genes. "GH" pituitary tumor cell lines constitute here also an excellent model. Not only do they possess the functional receptors for external signals, but they express tissue specific transcriptional factors such as Pit-1/GH-F1 necessary to the expression and optimal regulation of genes like PRL or GH genes. In this context, they offer advantageous model systems for investigating the regulations specific to the human GH and PRL genes (Morin et al., 1990; Peers et al., 1990) and thus could be considered for pharmacological screenings of drugs. ACKNOWLEDGMENTS These works were supported by the CNRS (UA1115) and grants from ARC (# 600084) and INSERM (CL # 874005). REFERENCES
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