Fibroblast Growth Factor Activation of the Rat PRL Promoter is ...

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Molecular Endocrinology 15(9):1517–1528 Copyright © 2001 by The Endocrine Society

Fibroblast Growth Factor Activation of the Rat PRL Promoter is Mediated by PKC␦ TWILA A. JACKSON, REBECCA E. SCHWEPPE, DAVID M. KOTERWAS, ANDREW P. BRADFORD

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Department of Obstetrics and Gynecology (T.A.J., D.M.K., A.P.B.), Department of Biochemistry and Molecular Genetics (R.E.S., A.P.B.), and the Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262 Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKC␦ is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKC␦ from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (␣, ␤I,

␤II, and ⑀) did not translocate in response to fibroblast growth factors. The PKC␦ subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKC␦ adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKC␦ isoform. PKC inhibitors selective for the conventional ␣ and ␤ isoforms or dominant negative PKC␣ adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKC␦ expression but not by an analogous dominant negative PKC␣ construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKC␦. (Molecular Endocrinology 15: 1517–1528, 2001)

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Several lines of evidence implicate FGFs in pituitary tumorigenesis. FGF-2 was originally identified in pituitary extracts and is abundant in pituitary cells (10). It increases PRL secretion from normal and cultured pituitary-adenoma cell lines (11, 12) and stimulates differentiation of PRL secreting pituitary lactotrophs (13). Furthermore, patients with multiple endocrine neoplasia type 1 exhibit elevated plasma levels of FGF-2 (14). The oncogene FGF-4 is also expressed in human prolactinomas (15) and activates both PRL transcription and secretion in rat GH4T2 pituitary cells (6). GH4T2 cells that stably express FGF-4 form highly aggressive and invasive tumors upon subcutaneous injection into rats (6). Finally, pituitary adenomas exhibit altered FGFR subtype and isoform expression (16). Thus, FGFs may play a critical role in the development and pathogenesis of pituitary prolactinomas, but the mechanism of action of these growth factors and the components of the FGF signaling pathway in pituitary cells remain to be elucidated. Regulation of the rat PRL (rPRL) promoter in GH4T2 rat pituitary tumor cells provides a physiologically relevant system in which to define and characterize mediators of FGF signaling and the role of FGFs in tumorigenesis. GH4T2 cells are neuroendocrine cells that express the phenotypic markers PRL and GH and

IBROBLAST GROWTH FACTORS (FGFs) are members of a family of polypeptides that play a critical role in development, embryogenesis, and angiogenesis. They are also important regulators of cell growth, differentiation, survival, and motility. Several FGFs have been identified as oncogenes (1–3) and are implicated in the formation and progression of tumors in a variety of tissues including pituitary, breast, prostate, and ovary (3–6). FGFs mediate their biological effects via a family of at least four distinct transmembrane tyrosine kinase receptors [FGF receptors (FGFRs) 1–4] (2, 7, 8), which exhibit overlapping recognition and redundant specificity (9). In common with other growth factors, binding of FGFs to their receptors results in receptor dimerization, activation of intrinsic tyrosine kinase activity, and receptor autophosphorylation (2, 8). Activation of FGFRs also results in increased tyrosine phosphorylation of a number of intracellular proteins; however, very little is known about the cellular substrates of FGFRs or the components of the FGF signaling pathway. Abbreviations: CMV ␤gal, Cytomegalovirus ␤-galactosidase; DAG, diacyl glycerol; DN, dominant negative; FGF, fibroblast growth factor; FGFR, FGF receptor; PS, phosphatidyl serine; rPRL, rat PRL; TPA, 12-O-tetradecanoylphorbol-13-ester.

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maintain normal hormonal and growth factor responses (17). Utilizing this system, we recently demonstrated that the FGF-2 and the FGF-4 signal to the rPRL promoter are independent of Ras and Raf-1 but act via the MAPK pathway (18). PKC is a family of serine/threonine kinases that has been implicated in the regulation of numerous signaling pathways, including many that are Ras-independent, but dependent on MAPK (19–22). To date, at least 11 PKC isoforms have been identified: the phosphatidyl serine (PS), diacyl glycerol (DAG), and Ca⫹2 -dependent conventional isoforms, which include PKC␣, -␤I, -␤II, and -␥; the Ca⫹2 -independent novel isoforms, PKC␦, -⑀, -␪, and -␩, which require PS and DAG; and the atypical isoforms ␨, ␮, and ␫, which require only PS for activation. Emerging data demonstrate that rather than cellular redundancy, these isoforms have distinct functions within the cell, including mitogenesis, apoptosis, glucose transport, gene expression, and secretion (23–30). In the present study, we have further characterized the FGF signal transduction pathway leading to induction of rPRL promoter activity in GH4T2 pituitary cells. We demonstrate a critical role for PKC␦ in FGF-mediated activation of PRL transcription and show that PKC␦ lies upstream of MAPK kinase (MEK1) and MAPK in the FGF signaling pathway. These results represent one of the first examples of a physiological role for a specific PKC isoform in the regulation of pituitary lactotroph-specific gene expression.

RESULTS The PKC Inhibitor, Calphostin C, Blocks FGF-2 and FGF-4 Activation of the rPRL Promoter FGF-2 and FGF-4 activate the rPRL promoter in GH4T2 pituitary cancer cells (18). We have previously shown that the FGF-4 signal to the rPRL promoter is independent of Ras and Raf-1, but requires MAPK (18). In several Ras-independent, MAPK-dependent signaling cascades, MAPK is coupled to the receptors via PKC. These include angiotensin II type 1 receptor signaling to the c-fos gene, growth stimulation of endothelial cells via vascular endothelial growth factor receptor signaling, and lysophosphatidic acid receptor signaling (20–22). To determine whether the MAPKdependent FGF signal to the rPRL utilizes PKC, we tested the ability of the broad spectrum PKC inhibitor, calphostin C, to block FGF-2 and FGF-4 activation of the rPRL promoter. GH4T2 pituitary cells were transiently transfected with a rPRL luciferase promoter (pA3–425 PRL luciferase), pretreated with 100 nM calphostin C, and stimulated with FGF-2 or FGF-4 as indicated. As seen in Fig. 1, pretreatment with the general PKC inhibitor, calphostin C, reduced basal transcription by 30%, while FGF-2 and FGF-4 stimulated the rPRL promoter 4.5- 5 fold above basal promoter activity. Pretreatment with 100 nM calphostin C

Jackson et al. • Fibroblast Growth Factor Signaling via PKC␦

Fig. 1. Calphostin C Blocks FGF-2 or FGF-4 Stimulation of the rPRL Promoter GH4T2 pituitary cells were cotransfected with 3 ␮g of pA3rPRL-425luc and 0.3 ␮g of pCMV ␤gal and serum starved for 16 h. Cells were pretreated with 100 nM calphostin C in the presence of light and then treated with 2 ng/ml FGF-2 or FGF-4 or diluent, as indicated, for 6 h before harvest. Luciferase activity was normalized to ␤gal activity, and the nonstimulated (NS) basal activity of pA3rPRL-425luc was set at 1. Results shown are representative of six experiments done in triplicate, and data are expressed as fold ⫾ SD of triplicate transfections.

completely abolished both the FGF-2 and FGF-4 induction of the rPRL promoter. Similar results were obtained with the broad-spectrum PKC inhibitor, bisindolylmaleimide GF 109203, and using GH3 and GH4C1 cell lines (data not shown). These results indicate that PKC is an important mediator of FGF-2 and FGF-4 signaling to the rPRL promoter in pituitary cells. Chronic 12-O-Tetradecanoylphorbol-13 Ester (TPA) Treatment to Down-Regulate PKC Prevents FGF-2 or FGF-4 Stimulation of the rPRL Promoter To confirm the role of PKC in FGF-2 and FGF-4 signaling to the rPRL promoter, GH4T2 cells were chronically treated (16 h) with the phorbol ester, TPA, which has been shown to deplete cells of PKC isoforms via proteolysis (33, 34). GH4T2 pituitary cells were transfected with the rPRL-luciferase reporter construct, serum starved overnight, and treated with 0, 100, or 1,000 nM TPA for 16 h. Cells were then stimulated acutely with FGF-2, FGF-4, or TPA as indicated. As shown in Fig. 2, chronic TPA treatment rendered the cells refractory to subsequent stimulation by either FGF-2 or FGF-4 as well as to acute TPA stimulation in a dose-dependent manner. The 5-fold FGF-2 and FGF-4 activation of the rPRL promoter was reduced to approximately 2-fold after 100 nM chronic TPA treatment and completely abolished by 1,000 nM chronic


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Fig. 3. GH4T2 Cells Express Detectable Levels of PKC␣, -␤I, -␤II, -␦, and -⑀ GH4T2 cells (1 ⫻ 107) were grown in full serum, harvested in 500 ı`l of RIPA buffer and resolved on a 10% SDS-polyacrylamide gel. Results of Western blot using 100 ␮g of total cellular protein are shown.

Fig. 2. Chronic Phorbol Ester Treatment Renders GH4T2 Cells Refractory to FGF-2 or FGF-4 Stimulation GH4T2 pituitary cells were cotransfected with 3 ␮g of pA3rPRL-425luc and 0.3 ␮g of pCMV ␤gal. After transfected cells plated down (⬃4 h), cells were treated with TPA at the indicated concentrations for 16 h and then stimulated with 2 ng/ml FGF-2 (squares), FGF-4 (open circles), or 100 nM TPA (solid circles) for 6 h. Cells were harvested and assayed as described in Fig. 1.

TPA treatment. It has been demonstrated that TPA activates the rPRL promoter in GH4T2 cells (35) and, therefore, as a control we show that the 4-fold TPAinduced activation is also blocked by chronic TPA pretreatment. These results provide further evidence that PKC is an essential signaling component mediating FGF activation of the rPRL promoter in GH4T2 pituitary cells. GH4T2 Cells Contain Five PKC Isotypes The PKC family of serine/threonine kinases is comprised of numerous isoforms. To date, at least 11 isotypes have been identified (for reviews see Refs. 36 and 37), including the conventional ␣, ␤I, ␤II, and ␥ isoforms, the novel ␦, ␪, ␩, and ⑀ isoforms, and the atypical ␫, ␮, and ␨ isoforms. The expression profile of PKC isoforms in GH4T2 cells has not been characterized. To determine which isoforms of PKC are present in GH4T2 pituitary cells, Western blot analyses of 100 ␮g of whole-cell extracts were performed. As shown in Fig. 3, five isoforms of PKC are expressed at detectable amounts in GH4T2 cells, including the Ca⫹⫹, DAG, and PS-dependent, conventional PKC␣, -␤I, and -␤II isoforms (lanes 1–3), and the Ca⫹⫹- and PSdependent novel PKC␦ and -⑀ isoforms (lanes 4 and 5). PKC␥, the novel PKC isoforms ␪ and ␩, and the atypical PKCs were not detected. As a control for antibody activity for PKC␥, -␪, -␩, -␫, -␮, and -␨ (the PKC isoforms that we did not detect in GH4T2 cell extracts), these isoforms were detected by Western blotting in

100 ␮g of extracts of rat brain or 3T3 cells using the indicated antibodies (data not shown). FGF-4 Induces Membrane Localization of PKC␦ Activation of PKC is often correlated with its movement from the cytosol to cell membranes (reviewed in Ref. 38). To test whether FGFs induced translocation of specific PKC isotypes, identified in GH4T2 pituitary cells, we stimulated the cells with FGFs and then performed subcellular fractionation followed by Western blotting of the cytosolic and Triton X-100 soluble membrane fractions (39). The results of a representative FGF-4 translocation experiment are shown in Fig. 4; similar results were obtained using FGF-2 (data not shown). Data from multiple experiments, analyzed by densitometry, are summarized in Table 1. FGF-4 did not induce translocation of the three conventional PKC isoforms (␣, ␤I, and ␤II), whereas TPA treatment caused substantial movement of these isoforms to particulate fractions (Fig. 4). A portion of both PKC␦ (30%) and PKC⑀ (44%) was present in the membrane fraction before FGF treatment. FGF induced a dramatic redistribution of PKC␦, resulting in more than 80% membrane localization. In contrast, the relative distribution of PKC⑀ did not change in response to FGF (Fig. 4 and Table 1). These results indicate that FGF treatment induces specific translocation of PKC␦ to the membrane, consistent with a role for PKC␦ in FGF signal transduction. The PKC␦-Selective Inhibitor, Rottlerin, Blocks the FGF-2 and FGF-4 Signal to the rPRL Promoter To corroborate the translocation studies, we used pharmacological isoform-selective PKC inhibitors. Go¨ 6976 has been shown to selectively block the classical PKC␣ and -␤ isoforms with an IC50 of 2.3–6.2 nM (40). As shown in Fig. 5A, pretreatment of GH4T2 cells with Go¨ 6976 slightly reduced basal activity of the rPRL

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activity by PKA catalytic subunit expression or forskolin treatment (data not shown). Thus, the effects of rottlerin are not due to nonspecific cellular toxicity. These results indicate that FGF activation of PRL gene expression is independent of the conventional ␣ and ␤ PKC isozymes. Moreover, in combination with the specific FGF-induced translocation of PKC␦, but not PKC⑀, the data clearly implicate PKC␦ as the primary isoform mediating the stimulatory effects of FGF-2 and FGF-4 on the rPRL promoter.

Fig. 4. The Novel PKC ␦ Isoform Is Translocated to the Membrane Fraction in Response to FGF-2 or FGF-4 GH4T2 cells (1 ⫻ 107) were serum starved for 16 h, stimulated with 2 ng/ml FGFs for 5 min, and harvested in lysis buffer A (see Materials and Methods). Cells were sonicated and pelleted at 70,000 ⫻ g for 1.5 h. Supernatants obtained were stored as cytosolic fractions, and the pellets were resuspended in lysis buffer B, sonicated, and saved as particulate/membrane fractions. Equal volumes of cytosolic fractions (C) and membrane fractions (M) were resolved by SDSPAGE, transferred to Immobilon P, and probed with the indicated antibodies. NS indicates no stimulation with FGFs.

Table 1. FGF-Induced PKC Isoform Translocation Control

FGF4

PKC

␣ ␤I ␤II ␦ ␧

C

M

C

M

96 ⫾ 2.1 100 ⫾ 0.7 99 ⫾ 0.7 70 ⫾ 8.9 56 ⫾ 5.6

4 ⫾ 2.1 0 ⫾ 0.7 1 ⫾ 0.7 30 ⫾ 8.9 44 ⫾ 5.6

96 ⫾ 2.8 99 ⫾ 1.4 97 ⫾ 0.7 19 ⫾ 5.1 53 ⫾ 5.7

4 ⫾ 2.8 1 ⫾ 1.4 3 ⫾ 0.7 81 ⫾ 5.1 47 ⫾ 5.7

The percent of indicated PKC isoform located in either the cytosolic (C) or membrane fraction (M) is shown. Western blots, such as that shown in Fig. 4, were analyzed by densitometric scanning using an AlphaImager 2000. Results are the mean ⫾ SD. n ⫽ 3 for ␣, ␤I, and ␤II and n ⫽ 4 for ␦ and ␧.

promoter, but had no effect on the fold activation by FGF-2 (2.5- to 3-fold) or FGF-4 (4- to 5-fold) at doses up to 1 ␮M. As a control, pretreatment of GH4T2 cells with 100 nM Go¨ 6976 significantly inhibited TPA stimulation of a c-fos promoter-luciferase construct, demonstrating that the inhibitor is functional in GH4T2 cells (data not shown). In contrast, the PKC␦-selective inhibitor rottlerin (IC50 3 ␮M) (41) abrogated FGF induction of the rPRL promoter (Fig. 5B). Both FGF-2 and FGF-4 responses were completely blocked by pretreatment with 10 ␮M rottlerin. Although rottlerin has also been shown to inhibit the novel PKC⑀, the IC50 is 100 ␮M, or 33-fold higher than that for PKC␦. The basal activity of the rPRL promoter was also reduced by approximately 40% in response to rottlerin (Fig. 5B). However, rottlerin had no effect on the induction of rPRL promoter

Expression of Dominant-Negative PKC␦ Attenuates FGF-2 or FGF-4 Activation of the rPRL Promoter To corroborate the pharmacological inhibitor and translocation data, we used adenoviral vectors that express either a PKC␦ dominant negative (DN) protein containing a K376-to-R mutation (DN PKC␦), or wildtype PKC␦, in our transient transfection system (Carpenter, L., and T. Biden, unpublished observation). Using a green fluorescent protein adenoviral expression vector, we first determined that a multiplicity of infection of 10 yielded approximately 90% infection of the GH4T2 cells (data not shown). Cells were infected with either wild-type adenoviral PKC␦ or DN PKC␦ construct and then transiently transfected with the rPRL-luciferase reporter construct. Infection with wildtype PKC␦ had no effect on basal or FGF-stimulated rPRL promoter activity (Fig. 6). Overexpression of wildtype PKC␦ did not enhance the FGF-2 or FGF-4 responses, suggesting that endogenous PKC␦ is not present in limiting quantities with respect to this signal transduction pathway. However, infection of the cells with DN PKC␦ completely blocked the approximately 3-fold FGF-2 or FGF-4 activation of the rPRL promoter. In contrast, stimulation of rPRL promoter activity by oncogenic V12 Ras was not affected by DN PKC␦ expression (Fig. 6). Cells infected with adenovirus encoding a DN PKC␣ construct (K368 to R) (2) retained rPRL promoter FGF responsiveness (data not shown), indicating that DN PKC␦ selectively blocked the FGF signaling pathway. To further test the physiological role of PKC␦ in FGF modulation of PRL gene expression, we tested the ability of DN PKC␦ to block transcription of the endogenous PRL gene using Northern blot analysis. GH4T2 cells were infected with the wild-type or DN PKC␦ adenoviral expression vectors described above or with DN PKC␣ as a control for isozyme specificity. Cells were then stimulated with FGF-2 or FGF-4, and total cellular RNA was probed with a radiolabeled PRL cDNA. Fig. 7A depicts a representative Northern blot. PRL mRNA levels from multiple experiments were normalized to glyceraldhyde-3-phosphate dehydrogenase mRNA and the results expressed as fold increase over basal (Fig. 7B). DN PKC␦ completely blocked the 2-fold FGF stimulation of endogenous rPRL mRNA expression. In contrast, neither wild-type PKC␦ nor DN PKC␣ affected FGF stimulation of the endogenous



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Fig. 5. The PKC␦-Selective Inhibitor, Rottlerin, but Not the Conventional PKC-Selective Inhibitor, Go¨ 6976, Blocks the FGF-2 or FGF-4 Signal to the rPRL Promoter GH4T2 pituitary cells were cotransfected with 3 ␮g of pA3rPRL-425luc and 0.3 ␮g of pCMV ␤gal. Sixteen hours after transfection, cells were pretreated with the inhibitors at the indicated concentrations for 30 min and then stimulated with either 2 ng/ml of FGF-2 or FGF-4. Six hours post treatment cells were harvested and assayed as described in Fig. 1. Results shown are representative of three experiments, and data are expressed as fold ⫾ SD of triplicate transfections. Panel A shows pretreatment with Go¨ 6976 followed by stimulation with FGF-2 or FGF-4, and panel B shows pretreatment with rottlerin followed by stimulation with FGF-2 or FGF-4 as indicated.

rPRL gene expression. Thus, FGF induction of endogenous PRL gene expression is also dependent upon PKC␦. MAPK Is Downstream of PKC in the FGF-2 and FGF-4 Signal Transduction Pathway to the rPRL Promoter We have previously shown, using DN or inhibitory expression plasmids, that the FGF-2 or -4 signal to the

rPRL promoter is independent of Ras/Raf-1, but mediated via MAPK (18). To further investigate the role of MAPK in FGF regulation of the rPRL promoter and in GH4T2 pituitary cells, we used the MEK1 inhibitor, PD 98059, which inhibits activation of MAPK (44). As shown in Fig. 8A, treatment with PD 98059 inhibits FGF-2 or FGF-4 induction of rPRL promoter activity in a dose-dependent manner. These results confirm the critical role of MAPK activation in FGF-2 and FGF-4 signaling to the rPRL promoter.



Fig. 6. DN PKC␦ Blocks FGF-2 or FGF-4 Stimulation of the rPRL Promoter GH4T2 pituitary cells were cotransfected with 3 ␮g of pA3rPRL-425luc and 0.3 ␮g of pCMV ␤gal and then infected with a wild-type or DN PKC␦ adenoviral expression vector for 16 h. Cells were harvested and assayed for luciferase activity (luciferase was normalized to ␤gal activity). Data are expressed as mean fold activation of three independent transfection experiments performed in triplicate.

Our previous work documented FGF-induced activation of MAPK in GH4T2 cells (18). Here we examine the role of PKCs in FGF activation of MAPK using an antibody specific for the phosphorylated, active form of MAPK (Fig. 8, B and C). As shown in Fig. 8B, down-regulation of PKC isoforms by chronic TPA treatment dramatically inhibits both FGF-2 and FGF-4 stimulation of MAPK phosphorylation and activation. As a control, chronic TPA treatment also inhibited acute TPA induction of MAPK phosphorylation. These results indicate that FGF activation of MAPK is dependent on PKC. To address the role of specific PKC isoforms in FGF-mediated MAPK activation, we used a panel of selective PKC inhibitors (Fig. 8C). The general PKC inhibitor calphostin C (lanes 4 and 5) and the PKC␦selective inhibitor, rottlerin (lanes 4 and 8) substantially reduced FGF-induced MAPK phosphorylation. However, Go¨ 6976, which selectively inhibits the classical PKCs (␣, ␤⌱, and ␤II), had no effect on the ability of FGFs to activate MAPK (lanes 4 and 6). The MEK1 inhibitor, PD 98059, which inhibits FGF activation of the rPRL promoter (Fig. 8A), also inhibited FGFinduced MAPK phosphorylation/activation (Fig. 8C, lanes 4 and 7). Taken together, these results (Fig. 8, A–C) indicate that FGF activation of MAPK and rPRL promoter activity are primarily mediated via PKC␦ and further suggest that PKC␦ is upstream of MAPK in the FGF rPRL signal transduction pathway.

DISCUSSION FGFs play a critical role in pituitary development and lactotroph physiology and have been implicated in the formation and progression of pituitary tumors. FGF-2 is highly expressed in normal pituitary, where it plays a role in PRL secretion (11, 12) and has recently been shown to initiate differentiation of GHFT lactotroph precursor cells (45). FGF-2 also induces pituitary transforming gene (pttg) expression, which coincides with early lactotrophic hyperplasia, angiogenesis, and prolactinoma development (46). In humans, many malignant pituitary tumors including prolactinomas express the FGF-4 (hst) oncogene, which results in aggressive prolactinoma formation in SCID mice (47). Despite this evidence that FGFs are important in normal and pathological pituitary functions, FGF signal transduction pathways have not been extensively characterized. We have previously identified FGF response elements in the rPRL promoter and shown that FGF induction of the rPRL promoter in GH4T2 cells is mediated via MAPK. However, in contrast to other systems, FGF activation of the rPRL transcription is independent of Ras and Raf-1 (18). In this report we show that PKC is required for FGF induction of the rPRL promoter and activation of MAPK. Furthermore, we identify the specific PKC isoform, PKC␦, as the primary mediator of the FGF signal. General inhibitors of PKC



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Fig. 7. DN PKC␦ Inhibits FGF Induction of Endogenous rPRL mRNA A, GH4T2 cells were infected with the indicated adenoviral PKC construct and subsequently treated ⫾ FGF-2 for 8 h. RNA was isolated and probed for rPRL message by Northern blotting as described in Materials and Methods. B, Northern blots were quantitated using a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA). PRL message was normalized to glyceraldhyde3-phosphate dehydrogenase mRNA and FGF responses expressed as fold increase over control levels. Data are means ⫾ SD of three to five experiments. *, P ⬍ 0.05; **, P ⬍ 0.005 by the paired t test.

or down-regulation of PKC by chronic TPA treatment blocks FGF induction of the rPRL promoter (Figs. 1 and 2). Use of the isotype-selective PKC inhibitors, Go¨ 6976 and rottlerin, which target the conventional PKCs (␣, ␤, ␥) and ␦, respectively, indicate that FGF activation of the rPRL promoter is dependent on the novel PKC␦ isoform (Fig. 5). Consistent with this hypothesis, FGFs translocate PKC␦, but not the conventional ␣, ␤, and ␥ or the novel ⑀ isoforms from soluble to particulate fractions, implying selective activation of PKC␦ in response to FGF treatment (Fig. 4 and Table 1). To confirm the role of PKC␦, we used adenoviral constructs encoding a kinase dead PKC␦ mutant (DN PKC␦), which functions as a specific DN inhibitor. Expression of DN PKC␦ abrogated activation of the rPRL promoter by FGF-2 and -4 (Fig. 6). Viral mediated expression of wild-type PKC␦ did not significantly potentiate the FGF response, implying that PKC␦ is not limiting in GH4T2 cells. Induction of exogenous rPRL promoter activity by oncogenic Ras was not affected

by DN PKC ␦ expression (Fig. 6), indicating a selective inhibition of the FGF signaling pathway and excluding nonspecific viral toxicity. The use of adenoviral vectors also allowed us to investigate the role of PKC␦ in FGF induction of the endogenous rPRL gene. Northern blot analysis of rPRL mRNA shows that DN PKC␦ completely blocked FGF induction of PRL transcription, whereas neither wild-type PKC ␦ nor DN PKC ␣ had an effect (Fig. 7). Thus, using a variety of experimental approaches, our results demonstrate a critical role for PKC␦ in transducing the FGF-inductive signal to the rPRL promoter. We have previously shown that FGF-2 and FGF-4 activate the rPRL promoter via MAPK but do not utilize Ras or Raf-1 as upstream activators of MAPK in the GH4T2 pituitary cell line. Treatment of GH4T2 cells with the MEK1 inhibitor PD 98059 completely inhibited FGF stimulation of rPRL promoter in a dose-dependent manner (Fig. 8A). These results suggest that the FGF signal is mediated via MEK1 activation of MAPK.



Fig. 8. FGF Stimulation of the rPRL Promoter Is Mediated by PKC␦-Dependent Activation of MAPK A, GH4T2 pituitary cells were cotransfected with 3 ␮g of pA3rPRL-425luc and 0.3 ␮g of pCMV ␤gal. Sixteen hours after transfection, cells were pretreated with PD98059 at the indicated concentrations for 30 min and then stimulated with either 2 ng/ml of FGF-2 or FGF-4. Six hours post treatment cells were harvested and assayed as described in Fig. 1. Results shown are representative of three experiments, and data are expressed as fold ⫾ SD of triplicate transfections. B, GH4T2 cells were treated for 16 h with 100 nM TPA (cTPA) and then stimulated acutely with TPA (100 nM) (acTPA), FGF-2 or FGF-4 (10 ng/ml). Ten minutes after stimulation, cells were harvested and protein concentration was determined. Fifty micrograms of total protein were analyzed by Western blotting using either a phospho-specific MAPK primary antibody or a pan MAPK antibody. Purified, nonphosphorylated MAPK (MK) and phosphorylated MAPK (pMK) were included as controls for antibody specificity. The pan MAPK panel is included as an equivalent loading control. C, Cells were pretreated with the general PKC inhibitor, calphostin C, the conventional PKC inhibitor, Go¨ 6976, MEK1 inhibitor, PD98059, or the PKC␦-selective inhibitor rottlerin for 30 min and then stimulated with 10 ng/ml FGF-4. Ten minutes after stimulation, cells were harvested and protein concentration was determined. Fifty micrograms of total protein were analyzed by Western blotting using either a phospho-specific MAPK primary antibody or a pan MAPK antibody.


PKC-dependent activation of MAPK has been documented in two other pituitary cell signaling systems— TRH in GH3 lactotrophs and GnRH in ␣T3–1 cells (42, 43, 48). In other cell types, PKC has been implicated in several signal transduction pathways that are Rasindependent yet coupled to MAPK. These include angiotensin II type 1 receptor signaling to the c-fos gene, growth stimulation of endothelial cells via vascular endothelial growth factor receptor signaling, and lysophosphatidic acid receptor signaling (20–22). FGFs stimulate MAPK phosphorylation and activation in GH4T2 cells (Fig. 8, B and C, and Ref. 18). Downregulation of PKCs blocked FGF activation of MAPK, placing PKC upstream of MAPK in the FGF signaling pathway (Fig. 8B). The PKC inhibitor, calphostin C or the PKC␦-selective antagonist, rottlerin, also abrogated FGF induction of MAPK (Fig. 8C). However, the conventional PKC (␣, ␤, and ␥) inhibitor Go¨ 6976 had no effect on FGF induction of MAPK. Thus, we have shown that FGF activation of the rPRL promoter is dependent upon both PKC␦ and MAPK. Moreover, since inhibitors of PKC␦ block FGF activation of MAPK, our results suggest that PKC␦ is upstream of MAPK in the FGF signal transduction pathway impacting on the rPRL promoter. The PKC family has long been implicated in the control of cellular functions and modulation of signal transduction in the hypothalamic pituitary axis, regulation of hormonal synthesis and secretion, and cell type ontogeny (49–51). Recent evidence suggests that the multiple PKC isoforms are not redundant but exert specific effects to up- or down-regulate cell growth, gene expression, cell differentiation, and apoptosis (21, 23, 52, 53). For example, PKC plays a key role in the GnRH stimulation of LH and FSH synthesis and secretion from pituitary gonadotropes (54). GnRH treatment of gonadotroph-derived ␣T3–1 cells results in differential up-regulation of PKC␦ and -⑀ mRNA levels with concomitant translocation of both PKC isoforms to the membrane (52). Multiple PKC isoforms are also involved in distinct aspects of the control of PRL synthesis and secretion. PKC⑀ has been implicated in TRH signaling and PRL secretion in pituitary cells (50, 55), whereas the effects of TRH on actin cytoskeletal reorganization are independent of PKC⑀ (27). TRH treatment of pituitary GH3B6 cells resulted in translocation of PKC␣ to regions of cell-cell contact (56). PKC⑀ is also involved in mediating the antiproliferative effects of dopamine (57), while increased PRL release from rat pituitary lactotrophs induced by dopamine withdrawal is associated with selective translocation and activation of PKCs ␣ and ␤ (51). Inhibition of PRL gene expression by TGF␤2 in GH3 cells is thought to be mediated by decreases in the activity of a select subset of PKC isozymes (58). Finally, in this report, we show that PKC␦ is a critical component of the FGF signaling pathway leading to induction of PRL gene expression. Thus, differential activation of functionally distinct PKC isoforms, such as PKC␦, in response to specific


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signal transduction pathways, e.g. FGF, provides a mechanism to coordinate and integrate both inductive and inhibitory stimuli regulating pituitary hormone synthesis and secretion and gene expression.

MATERIALS AND METHODS Cell Culture and Transfections GH4T2 rat pituitary tumor cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 12.5% horse serum and 2.5% FCS (referred to as full serum) (Life Technologies, Inc.) and 50 ␮g/ml penicillin and streptomycin (Life Technologies, Inc.). Cells were maintained at 37 C in 5% CO2. Transient transfections were carried out by electroporation, as described previously (31, 32). Briefly, media were changed 4–12 h before each transfection, and cells were harvested at 50–70% confluency and electroporated in full serum as described (32). After electroporation, 200 ␮l cells (3–5 ⫻ 106) were plated in 3 ml DMEM without serum for a final concentration of 0.94% serum to achieve low levels of endogenous growth factors. Cells were incubated for 1624 h and treated with FGF-2 or FGF-4 (R & D Systems, Minneapolis, MN), or diluent (0.1% BSA in PBS) at a final concentration of 2 ng/ml. FGF responses were assayed 6 h after treatment. Electroporations were performed in triplicate for each condition within a single experiment, and experiments were repeated using different plasmid preparations of each construct at least three times. Luciferase and ␤-galactosidase assays were performed as previously described (31, 32). Adenoviral infections were performed as follows: adenoviruses were added to cells in one-third volume of normal media with full serum at a multiplicity of infection of 10–20. Plates were shaken every 10 min for 1 h after which media containing full serum were added to regular volume. Cells were incubated for 24 h, harvested, and used for electroporation as described above. Adenoviral constructs were a generous gift of Drs. Lee Carpenter and Trevor Biden (Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia). Pharmacological Reagents Calphostin C, Go¨ 6976, rottlerin, and TPA were obtained from Calbiochem (San Diego, CA), and PD98059 was obtained from New England Biolabs, Inc. (Beverly, MA). All pharmacological reagents were prepared and stored according to the manufacturer’s specifications and used at the concentrations indicated in the specific experiments. Calphostin C pretreatment was 30 min to 1 h in the presence of light and Go¨ 6976, PD98059, and rottlerin pretreatments were 30 min to 1 h at 37 C. For PKC down-regulation studies, cells were treated with TPA for 16 h (chronic TPA treatment). Plasmid Constructs The promoter construct pA3 -425 rPRLluc and pCMV ␤gal (cytomegalovirus ␤galactosidase) (CLONTECH Laboratories, Inc., Palo Alto, CA) have been described previously (18). Membrane Localization Assay GH4T2 cells were serum starved (0% serum) for 24 h and subsequently stimulated with FGFs or TPA for the indicated times. Cells were washed once with ice-cold PBS and har-


vested in lysis buffer A (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1% ␤-mercaptoethanol and 1⫻ Complete Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Indianapolis, IL). Cells were sonicated for 10 sec, output 4 on constant duty cycle, using a Branson Sonicator (Branson Ultrasonics Corp., Danburg, CT) and pelleted at 70,000 ⫻ g for 1.5 h. The supernatant was collected as the cytosolic fraction. The pellet was resuspended in lysis buffer B (lysis buffer A ⫹ 1% Triton X-100) by 10 sec sonication and repelleted for 15 min at 13,000 ⫻ g in a microfuge. The supernatant was collected as the Tritonsoluble particulate fraction. Western Blot Analysis GH4T2 cells were serum starved overnight and treated with 2 ng/ml FGF-2 or FGF-4 or the equivalent volume of diluent for the indicated times. Cells (107) were washed in cold PBS and harvested in 500 ␮l RIPA buffer [PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Complete Protease Inhibitor Cocktail (Roche Molecular Biochemicals)]. Equal amounts of protein (50–100 ␮g), as determined by the Pierce Mini BCA protein assay (Pierce Chemical Co., Rockford, IL), were resolved by electrophoresis on 10% polyacrylamide-SDS gels and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA) according to the manufacturer’s protocol. Membranes were blocked 1 h at room temperature or overnight at 4 C in blocking buffer [5% nonfat dry milk in TBS ⫹ 0.1% Tween 20]. They were then incubated with primary antibodies directed against various isoform-specific epitopes of PKC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to the manufacturer’s protocol (1:500 in blocking buffer, 1 h at room temperature). For Western blot analyses using MAPK phospho-specific or pan primary antibodies (New England Biolabs, Inc.) the primary antibody incubations were done for 20 h at 4 C. As a control for phospho- and nonphospho-MAPK antibody specificity, 20 ng of bacterially expressed phospho- or nonphospho-MAPK 2 were included (New England Biolabs). Membranes were incubated with a horseradish peroxidase-conjugated, goat antirabbit secondary antibody (Life Technologies, Inc.) diluted 1:5,000 in blocking buffer for 1 h at room temperature or overnight at 4 C. Protein was detected using the Super Signal chemiluminescence assay (Pierce Chemical Co.) according to the manufacturer’s protocol. Where indicated, membranes were stripped of antibody for 30 min at 50 C according to the Super Signal protocol and reprobed as described above. Northern Blot Analysis GH4T2 cells were serum starved for 24 h and stimulated with 10 ng/ml FGF-2 or FGF-4 for 8 h. Cells were then resuspended in RNA-STAT60 (Tel-Test, Inc., Friendswood, TX), and RNA was prepared as per manufacturer’s protocol. Ten micrograms of RNA were electrophoresed on a 1.4% agarose/1.75% formaldehyde 3-(N-morpholino)propanesulfonic acid gel and transferred to a Hybond N⫹ membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). RNA was cross-linked to the filter using 50 mM NaOH for 5 min and 2⫻ SSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) for 5 min. Blots were prehybridized 1 h at 42 C in Ultrahyb (Ambion, Inc., Austin, TX) and then probed overnight at 42 C with a labeled full-length rPRL probe (5 ⫻ 106 cpm/blot) in Ultrahyb hybridization solution. Blots were washed three times with 0.2⫻ SSC, 0.5% SDS at 65 C to remove background signal followed by autoradiography.

Acknowledgments We thank Drs. Carpenter and Biden for PKC adenoviral constructs and Dr. J. J. Tentler and J. D. Graham for assis-


tance with Northern blots. We also thank Drs. D. F. Gordon, A. Gutierrez-Hartmann, M. E. Reyland, J. J. Tentler, and W. M. Wood for critical reading and discussion of this manuscript.

Received October 25, 2000. Accepted May 16, 2001. Address requests for reprints to: Dr. Twila A. Jackson, Department of Obstetrics/Gynecology, Colorado Cancer Center, University of Colorado Health Science Center, 4200 East Ninth Avenue, Box B-198, Denver, Colorado 80262. E-mail: [email protected]. This work was supported by NIH Grant DK-53496 (to A.P.B.). T.A.J. is supported by National Research Service Award DK-10031.

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15th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology Recent Advances in Steroid Biochemistry & Molecular Biology May 17–20, 2002 — Munich (Mu¨ nchen), Germany The 15th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology—Recent Advances in Steroid Biochemistry & Molecular Biology—will be held in Munich (Mu¨ nchen), Germany on May 17–20, 2002. The following topics will be considered: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Steroid Receptors, Structure, Gene Expression, and Mechanism of Action Non Genomic Effect of Steroid Hormones Steroid Membrane Receptors Steroids and Cancer Steroids in the Central and Peripheral Nervous Systems Steroids and the Menopause Enzyme Modulators Steroid Hormones, Phyto-, Xeno-Estrogens, and the Environment Steroids and Sport

Lectures (approximately 30–35) will be by invitation of the Scientific Organizing Committee and, in addition, there will be poster sections. All poster presentations will be subject to selection by the Scientific Organizing Committee. Abstracts (maximum 200 words) must be sent to Prof. J. R. Pasqualini by Monday, February 4th, 2002 (postmark) (Original ⫹ 12 copies). General Scientific Secretariat: Prof. J. R. Pasqualini, Steroid Hormone Research Unit, Institut de Pue´ riculture, 26 Boulevard Brune, 75014 Paris, France. Tel.: (33-1)⫹45 39 91 09/ 45 42 41 21; Fax: (33-1)⫹45 42 61 21; e-mail: [email protected] Local Organizing Committee: Prof. H. Michna, Technical University of Munich, Olympiapark, Conollystr. 32, D-80809 Mu¨ nchen, Germany. Tel: (49)⫹89/289-24601; Fax: (49)⫹89/289-24636; e-mail: [email protected]