The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth
2007; Springer Nature; Volume: 26; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601636
ISSN1460-2075
AutoresCarmen Cañibano, Noela Rodríguez-Losada, Carmen Sáez, Sulay Tovar, Montserrat García-Lavandeira, Maria Grazia Borrello, Anxo Vidal, Frank Costantini, Miguel Á. Japón, Carlos Diéguez, Clara V. Álvarez,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoArticle22 March 2007free access The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth Carmen Cañibano Carmen Cañibano Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Noela L Rodriguez Noela L Rodriguez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Carmen Saez Carmen Saez Department of Pathology, Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Sulay Tovar Sulay Tovar Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Montse Garcia-Lavandeira Montse Garcia-Lavandeira Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Maria Grazia Borrello Maria Grazia Borrello Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy Search for more papers by this author Anxo Vidal Anxo Vidal Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Frank Costantini Frank Costantini Department of Genetics and Development, Columbia University Medical Center, New York, USA Search for more papers by this author Miguel Japon Miguel Japon Department of Pathology, Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Carlos Dieguez Carlos Dieguez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Clara V Alvarez Corresponding Author Clara V Alvarez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Carmen Cañibano Carmen Cañibano Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Noela L Rodriguez Noela L Rodriguez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Carmen Saez Carmen Saez Department of Pathology, Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Sulay Tovar Sulay Tovar Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Montse Garcia-Lavandeira Montse Garcia-Lavandeira Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Maria Grazia Borrello Maria Grazia Borrello Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy Search for more papers by this author Anxo Vidal Anxo Vidal Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Frank Costantini Frank Costantini Department of Genetics and Development, Columbia University Medical Center, New York, USA Search for more papers by this author Miguel Japon Miguel Japon Department of Pathology, Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Carlos Dieguez Carlos Dieguez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Clara V Alvarez Corresponding Author Clara V Alvarez Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Search for more papers by this author Author Information Carmen Cañibano1, Noela L Rodriguez1, Carmen Saez2, Sulay Tovar1, Montse Garcia-Lavandeira1, Maria Grazia Borrello3, Anxo Vidal1, Frank Costantini4, Miguel Japon2, Carlos Dieguez1 and Clara V Alvarez 1 1Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain 2Department of Pathology, Hospital Universitario Virgen del Rocio, Seville, Spain 3Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy 4Department of Genetics and Development, Columbia University Medical Center, New York, USA *Corresponding author. Department of Physiology, School of Medicine, University of Santiago de Compostela, c/San Francisco s/n, 15782 Santiago de Compostela, Spain. Tel.: +34981582658; Fax: +34981574145; E-mail: [email protected] The EMBO Journal (2007)26:2015-2028https://doi.org/10.1038/sj.emboj.7601636 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Somatotrophs are the only pituitary cells that express Ret, GFRα1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit-1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit-1 siRNA. The Pit-1 overexpression is mediated by sustained activation of PKCδ, JNK, c/EBPα and CREB induced by a complex of Ret, caspase 3 and PKCδ. In the presence of GDNF, Akt is activated, and the Pit-1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit-1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit-1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit-1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas. Introduction Final adult height and body composition are maintained in a constant interval for each given species. Somatototrophs, the pituitary cells secreting growth hormone (GH), are specialized secretory cells. However, they are not terminally differentiated: the pituitary retains some plasticity throughout life, enabling appropriate responses to events such as puberty and pregnancy (Melmed, 2003). Somatotrophs play a key role in the control of growth during infancy and puberty, although they continue to secrete GH throughout adult life. Somatotroph number and function are tightly regulated, as GH hypersecretion leads to excessive growth in the form of either gigantism or acromegaly. Genetic factors leading to somatotroph hypoplasia are well known, but little is known about the mechanisms controlling physiological adjustments in somatotroph number. In this connection, the incidence of pituitary somatotroph adenomas is surprisingly low (about 3–4 cases per 106 per year) compared with adenomas of other endocrine glands (Holdaway and Rajasoorya, 1999). More puzzling still is the consistent benignity of these tumors, which never metastasize (Melmed, 2003; Kaltsas et al, 2005). We have recently demonstrated that somatotrophs are the only pituitary secretory cells expressing Ret, GDNF and GFRα1, both in humans (Japon et al, 2002) and in rats (Urbano et al, 2000). This expression pattern is maintained in all somatotroph adenomas. Ret is a transmembrane receptor well known for its differentiation- and survival-controlling functions in epithelial cells, neurons and neuroendocrine cells. Alternative splicing produces two main isoforms: Ret long (Ret-L or Ret51), 1072 amino acids (aa) long, and Ret short (Ret-S or Ret9), 1114 aa long, with different sequences in the C-terminal region (51 aa long in Ret-L and 9 aa long in Ret-S) (Tahira et al, 1990). Ret has four coreceptors (GFRα1, 2, 3 and 4), with ligands GDNF, NTN, ART and PSP, respectively. Extensive research on Ret function in mammals indicates that it has two main functions. First, it actively promotes survival, growth and extension/migration of cells such as neurons (neurite and axonal sprouting) and epithelial renal cells (branching). In these roles, Ret binds to its ligand GDNF and its GPI-anchored extracellular coreceptor GFRα1, and this binding induces the tyrosine kinase activity of Ret, triggering various signal transduction pathways (Bredesen et al, 2004; Porter and Dhakshinamoorthy, 2004; Arighi et al, 2005). Second, Ret appears to induce apoptosis, but only in the absence of GDNF; to date, however, this effect has only been demonstrated in transient transfection of HEK293T and neuroblastoma cell lines (Bordeaux et al, 2000). In line with this, Ret has been included in the family of 'dependence receptors', together with p75NTR, DCC, UNC5H, PTC1 and the androgen receptor. In humans, mutations that activate Ret lead to multiple endocrine neoplasia type 2 (MEN 2), whereas inactivating mutations lead to Hirschprung's disease (Santoro et al, 2004; Arighi et al, 2005). Here, we report a study of Ret functions in a somatotroph cell line and in primary pituitary cultures. We uncover a pathway in which Ret, in the absence of GDNF, induces Pit-1 overexpression, leading to increased p53 expression and apoptosis. We identify the kinases involved in the induction of Pit-1 promoter overexpression by Ret. The cytoplasmic Ret portion (IC-Ret) complexes with caspase 3 and the kinase PKC delta, all becoming cleaved during the process. We also present results indicating that newborn Ret KO mice pituitaries are larger than normal, showing Pit-1-expressing cell and somatotroph hyperplasia. Finally, the Ret/Pit-1/p53 apoptotic pathway acts in an in vivo model of pituitary hyperplasia. In this model, retroviral Ret delivery suppressed estrogen-induced hyperplasia but did not alter normal pituitary function. Results Ret-induced apoptosis is associated with Pit-1 expression To investigate the role of Ret in somatotrophs, we used a pituitary cell line, GH4C1, that does not express some of the characteristic receptors of normal somatotrophs (e.g. GHRH-R), but that nonetheless expresses the ghrelin receptor (NRL unpublished) and small amounts of Pit-1, and secretes some GH. This cell line does not express Ret but expresses both GFRα1 and GDNF mRNA (data not shown) and the corresponding proteins (Figure 1A, left). We tried to produce transfectants stably expressing human Ret-L or Ret-S; colonies were obtained in various transfections, but in no case was Ret expression detected (Figure 1A). While transient transfection of GH4C1 or human embryonic kidney (HEK293) cells led to Ret expression (Figure 1A), Ret stable transfection had a deleterious effect on colony number after selection with neomycin (G418) (Supplementary Figure 1). In a previous study using the HEK293T cell line, transient transfection with Ret induced apoptosis that could be blocked by GDNF (Bordeaux et al, 2000). Similarly, in our somatotroph cells, transfection with Ret (either isoform) potently induced apoptosis within 24 h, whereas addition of GDNF (50 ng/ml) completely blocked this response (Figure 1B, left). zVAD, a broad caspase inhibitor, likewise blocked the apoptosis, suggesting an involvement of caspase activity (Figure 1B, right). Figure 1.Both the Ret-L and Ret-S isoforms strongly induced apoptosis and Pit-1 expression in the GH4C1 somatotroph line. (A) GH4C1 cells expressed GDNF and GFRα1 but not Ret. No stable Ret-expressing transfectants could be obtained (results shown only for Ret-L): the few colonies obtained were all Ret-negative, except colony 6B, which died after the second passage. In transient transfections, full-length Ret (150–170 kDa) was expressed strongly in HEK293 cells and weakly in GH4C1 cells. (B) Forty-eight hours after transient transfection of GH4C1 cells with either Ret-L or Ret-S, marked apoptosis was detected. Apoptosis could be prevented by treatment with GDNF or the broad caspase inhibitor zVAD. (C) In the absence of Ret, GDNF had a weak inhibitory effect on Pit-1 mRNA expression in GH4C1 cells. (D) Transfection with Ret induced a marked increase in Pit-1 mRNA expression, which was blocked by GDNF. (E) The broad caspase inhibitor zVAD blocked the effect of Ret on Pit-1 mRNA expression. (F) Ret transfection likewise induced a marked increase in Pit-1 protein expression, again blocked by GDNF (results shown only for Ret-L). Note that detection of full-length Ret required immunoprecipitation or loading more than 150 μg proteins/lane, whereas the processed intracellular fragment (IC-Ret) could be detected easily with 50 μg proteins/lane. Both IC-Ret and activated caspase 3 (19 kDa) were induced by transfection with Ret when GDNF was absent but not when GDNF was present. (G) zVAD also reduced the Ret-induced increase in Pit-1 protein expression (results shown only for Ret-S) and blocked IC-Ret formation and activation of caspase 3. Download figure Download PowerPoint We next investigated the effects of Ret transfection and GDNF on the expression of specific pituitary somatotroph genes. In the absence of Ret, GDNF had no consistent effect on the hormones GH or PRL (data not shown), but had a small dose-dependent inhibitory effect on Pit-1 mRNA expression (Figure 1C). Transfection with Ret induced a marked increase in Pit-1 mRNA, which was blocked by incubation with GDNF (Figure 1D). The caspase inhibitor zVAD, which blocked Ret-induced apoptosis, also blocked the effect of Ret on Pit-1 mRNA expression (Figure 1E). Ret likewise induced an increase in Pit-1 protein levels, which were again reduced by GDNF and zVAD (Figure 1F and G). Ret has been classed as a 'dependence receptor': in HEK and neuroblastoma cell lines, it actively induces apoptosis in the absence of its ligand GDNF, whereas the presence of the ligand allows cell survival (Bordeaux et al, 2000; Bredesen et al, 2004; Porter and Dhakshinamoorthy, 2004). The Ret protein is processed by caspase 3 at two intracytoplasmic sites, releasing a fragment of about 40–50 kDa (IC-Ret) that has been proposed to induce apoptosis. IC-Ret was readily detected by Western blotting in our GH4C1 cells after transient transfection with either Ret-L or Ret-S. IC-Ret was not detectable in cells treated with zVAD, and levels were significantly reduced by GDNF (Figure 1F and G), in contrast with the previous findings in HEK cells. Basal levels of activated caspase 3 were present in the GH4C1 cells, and caspase-3 activity was strongly induced by Ret transfection and reduced by GDNF or zVAD; that is, the effects on caspase 3 paralleled the effects on apoptosis, IC-Ret presence and Pit-1 level (Figure 1F, G and B, E). In the presence of GDNF, Ret forms a heterodimeric complex with GDNF and GFRα1, activating Ret's tyrosine kinase activity (so that it phosphorylates susceptible cytoplasmic substrates). We therefore investigated whether the tyrosine kinase activity of Ret is necessary for induction of apoptosis and/or of Pit-1 expression. The kinase-dead mutants of Ret-L (Ret-LKR, K758R substitution) and Ret-S (Ret-SKR, K758R substitution) were transiently transfected into GH4C1 cells. The transfected cells did not show increased tyrosine kinase activity in the presence of GDNF (unlike cells transfected with Ret-L or Ret-S) (Figure 2B), although Ret was expressed to similar levels (Figure 2C). While all Ret isoforms had apoptotic effects, the protective effects of GDNF were not seen in the cells transfected with the kinase-dead mutants (Figure 2D). Interestingly, both kinase-dead Ret isoforms strongly induced Pit-1 mRNA and protein expression (like the normal isoforms), but, unlike in cells transfected with the normal isoforms, GDNF did not inhibit this response (Figure 2E and F); again, this is in line with a relationship between Ret's apoptotic activity and its induction of Pit-1 expression. Figure 2.Ret-induced apoptosis and Pit-1 expression are independent of Ret's tyrosine kinase activity, but dependent on activation of caspase 3. (A) Cartoon showing the wild-type and mutant receptors used in these experiments. Ret-LKR is a kinase-dead mutant (K758R) of Ret-L; Ret-SKR is a kinase-dead mutant (K758R) of Ret-S; Ret-SDN, cloned in the pBR expression vector, has a substitution in the proximal caspase-3-processing consensus site of the Ret-S isoform (control: Ret-Swt in the same vector). (B) When expressed in HEK293 cells, neither kinase-dead mutant showed tyrosine kinase activity. As HEK293 cells secrete GDNF, a basal phosphorylation of the Ret receptor is seen in the untreated lanes. (C) The kinase-dead mutants were expressed in GH4C1 somatotrophs and (D) strongly induced apoptosis that could not be blocked by GDNF, like the wild-type isoforms. The kinase-dead mutants likewise markedly increased Pit-1 mRNA (E) and protein (F) expression; however, these increases were not blocked by GDNF. (G) In cells transfected with the Ret-SDN mutant, neither IC-Ret (the processed intracellular form) nor activated caspase 3 could be detected. In line with this, Ret-SDN did not induce either apoptosis (H) or Pit-1 mRNA (I), or protein (J) expression (*P<0.05; **P<0.01; ***P<0.001). Download figure Download PowerPoint Given our results showing that Ret's apoptotic activity is associated with caspase-3 processing and the appearance of the intracytoplasmic fragment IC-Ret, we transfected cells with Ret-SDN, a Ret-S mutant with a D707N substitution affecting the first caspase-3 consensus site in the cytoplasmic tail of the receptor (Figure 2A). As previously demonstrated (Bordeaux et al, 2000), transfected Ret-SDN could not be processed by caspase 3 in vivo, and no IC-Ret band was detected in Western blotting (Figure 2G). Ret-SDN did not induce either apoptosis (Figure 2H) or Pit-1 mRNA or protein expression (Figure 2I and J). Ret-induced apoptosis is mediated by Pit-1, and Pit-1 overexpression increases p53 levels The data shown in Figures 1 and 2 suggested a direct relationship between caspase-3-processed intracytoplasmic Ret, Pit-1 expression and apoptosis. The time course of caspase-3 activity in extracts of transfected cells showed that the maximal activity was reached immediately after initiating the Ret transfection (2 h) (Figure 3A). Coincidentally, the induction of Pit-1 expression in somatotrophs was a very early event, seen as soon as 2 h after Ret transfection (Figure 3B), and maintained throughout the experiment (Figure 3C). Ret also induced a marked increase in p53 at 24 h after transfection (Figure 3D) before the induction of apoptosis, which peaked around 48 h after transfection (see Figures 1 and 2). Incubation with zVAD (which blocked IC-Ret generation, induction of Pit-1 expression and apoptosis; Figures 1B, E and G) also blocked the p53 increase (Figure 3E). Figure 3.Ret-induced caspase-3 activation and Pit-1 overexpression in somatotrophs are early events causing a later p53 increase and apoptosis. (A) Time-course assay of caspase-3 activity after initiation of Ret-S transfection compared with empty-vector transfected cells or in the presence of the caspase-3 inhibitor DEVDfmk. The highest activity was seen at the first time point, only 2 h after the initiation of Ret transfection. (B) The Ret-induced increase in Pit-1 mRNA expression was already detectable within 2 h of transfection, suggesting a direct effect on the Pit-1 promoter that increased Pit-1 protein (C) with time after transfection. (D) An increase in p53 protein expression was only detectable 24 h after transfection. (E) Caspase inhibition with zVAD (which inhibited caspase-3 activity, Ret processing, apoptosis and Pit-1 overexpression; Figures 1 and 2) blocked the Ret-induced p53 increase. (F) Transfection with Pit-1 induced similar levels of apoptosis in somatotrophs as transfection with Ret; the two effects were not additive. (G) Intriguingly, Pit-1-induced apoptosis was also inhibited by GDNF (Supplementary Figure 2A). (H) Pit-1 siRNA blocked Pit-1 mRNA expression, whether basal or induced by Ret or Pit-1 transfection. For each Pit-1 siRNA treatment (+) and its control (−, GFP siRNA), lanes are shown with different exposure times. Pit-1 siRNA blocked apoptosis induced by Ret-L, Ret-S and Pit-1 (I) and the Ret-induced increase in Pit-1 and p53 protein expression (J). Download figure Download PowerPoint This sequence of events suggested that Ret-induced apoptosis in somatotrophs is mediated by Pit-1 induction. Transfection of a Pit-1 expression construct induced similar levels of apoptosis to Ret transfection, and the two treatments were not additive (Figure 3F). However, Pit-1-induced apoptosis was blocked by GDNF (Figure 3G), simultaneously with the inhibition of transfected Pit-1 expression (Supplementary Figure 2A). To confirm that Pit-1 expression is essential for Ret-induced apoptosis, we used Pit-1 siRNA to block basal and Ret-induced Pit-1 expression (Figure 3H). The presence of Pit-1 siRNA entirely blocked the apoptosis induced by transfection with Ret-L, Ret-S or Pit-1 (Figure 3I), and also blocked p53 induction (Figure 3J). These results suggest that caspase-3-processed IC-Ret induces Pit-1 expression, which in turn increases p53 expression and causes apoptosis. Ret-induced c/EBPα binding to the Pit-1 promoter depends on PKCδ, JNK and CREB Next, we performed experiments designed to identify kinases involved in the induction of Pit-1 expression by Ret. Sustained p-JNK and p-CREB expressions were detected in somatotrophs transfected with all Ret isoforms and mutants that induced apoptosis, but not in somatotrophs transfected with the non-apoptosis-inducing mutant Ret-SDN (Figure 4A). As previously described (Chiariello et al, 1998; Feng et al, 1999; Poteryaev et al, 1999; Trupp et al, 1999; Hayashi et al, 2000; Pezeshki et al, 2001), short-term (15 min) treatment with GDNF also induced p-JNK or p-CREB activation (Figure 4B) that returned to basal levels after 30 min (Supplementary Figure 2B) and was not altered by Ret expression. However, long-term (24 h) treatment with GDNF, which blocks Ret-induced apoptosis, also blocked the Ret-induced p-JNK and p-CREB sustained expression (Figure 4C). Likewise, we investigated whether PKCδ is activated by Ret. PKCδ has been implicated in apoptosis induced by DNA-damaging agents or Fas ligand: after its induction, PKCδ is phosphorylated (p-PKCδ) and processed, and the free catalytic domain migrates to the nucleus inducing apoptosis (DeVries et al, 2002; Mecklenbrauker et al, 2002, 2004), although the mechanism remains unknown. p-PKCδ was detected in somatotrophs transfected with the apoptosis-inducing Ret forms, but not in cells transfected with Ret-SDN (Figure 4D). In contrast to the effects on JNK or CREB, short-term treatment (15 min) with GDNF reduced p-PKCδ levels in cells transfected with Ret-L and Ret-S but not in cells transfected with the kinase-dead mutant Ret-LKR (Figure 4D). This reduction in p-PKCδ levels by GDNF was maintained after 24 h of treatment, coinciding with the reduction in p-CREB and p-JNK levels (Figure 4C). Ret-induced p-PKCδ production was also blocked by the caspase inhibitor zVAD (Figure 4E). These results suggested that Ret may induce Pit-1 and apoptosis through activation of PKCδ. Rottlerin, a PKCδ inhibitor, blocked PKCδ phosphorylation by all the apoptosis-inducing Ret forms (Figure 4F). Rottlerin also blocked Ret-induced p-JNK expression but not Ret-induced p-CREB expression (Figure 4G). Rottlerin strongly reduced Ret-dependent Pit-1 expression, and in line with this reduced Ret-induced apoptosis; however, it did not affect Pit-induced apoptosis (Figure 4H and I). Finally, killer CREB, a dominant-negative form of CREB, significantly reduced Ret-induced apoptosis (Figure 4H). These data suggest a two-branched pathway for Ret-induced Pit-1 expression, one branch dependent on activation of PKCδ/JNK and the other dependent on activation of CREB. Pit-1 mRNA induction by this pathway was very rapid, suggesting a transcriptional effect. The most important consensus binding sites on the rat Pit-1 promoter are shown in Figure 4J. Both CRE elements are essential for GHRH- and ghrelin-induced Pit-1 expression (Soto et al, 1995; Garcia et al, 2001). In somatotrophs cotransfected with Ret and promoter/Luc constructs with decreasing promoter length, luciferase activity was detected with the −2500, −959 and −358 promoter fragments (strongest activity with the −358 fragment), whereas further shortening to −231 abolished the Ret-dependent activity (Figure 4J). Luciferase activity was also completely abolished by treatment with GDNF or by cotransfection with Ret-SDN instead of Ret-S (Figure 4K). Luciferase activity in cells cotransfected with Ret-S and the −358 promoter fragment was markedly reduced by rottlerin and by killer CREB, and completely abolished by adding both together, confirming the additive effects of the PKCδ/JNK and CREB pathways (Figure 4L). These results indicate that the Ret-responsive region of the rat Pit-1 promoter lies between this protein's positions −358 and −231. A consensus binding site for the Pit-1 transcription factor c/EBPα appears near the first CRE, just before −231. Interestingly, the human Pit-1 promoter has a similar conserved site located upstream to the TATA region (Pfaffle et al, 1992), and initial results corroborate a Ret-dependent regulation of the hPit-1 promoter (data not shown). To confirm that Ret induces binding of c/EBPα to the rat Pit-1 promoter, we used chromatin immunoprecipitation (ChIP) assay with c/EBPα-specific antibodies, followed by PCR using primers for a small region of the Pit-1 promoter, including the c/EBPα binding site. A basal level of c/EBPα binding was detected in untransfected cells, and an increase in this binding in Ret-transfected cells was detected within only 4 h of transfection. The increase in c/EBPα binding was completely abolished by GDNF and significantly reduced by rottlerin or cotransfection with killer CREB (Figure 4M). Figure 4.Ret-induced sustained activation of PKCδ, JNK and CREB leads to binding of c/EBPα to the Pit-1 promoter. (A) Twenty-four hours after transfection of GH4C1 cells with apoptosis-inducing Ret forms, but not after transfection with Ret-SDN, p-JNK and p-CREB levels showed a sustained increase. (B) We could not study the short-term effects of GDNF on p-JNK or p-CREB levels, as GDNF induced short-term phosphorylation of both kinases in the absence of Ret (peak at 15 min, return to basal at 30 min; Supplementary Figure 2B). (C) Long-term treatment with GDNF (24 h) blocked the Ret-induced phosphorylation of both kinases. (D) Transfection of GH4C1 cells with the apoptosis-inducing Ret forms, but not Ret-SDN, induced a sustained increase in levels of p-PKCδ. Brief treatment with GDNF (15 min) inhibited this PKCδ phosphorylation, which was maintained after long-term (24 h) GDNF treatment (C). (E) zVAD treatment blocked the Ret-induced phosphorylation of PKCδ. (F) Rottlerin (Rott), a specific PKCδ inhibitor, effectively blocked the Ret-induced phosphorylation of PKCδ and (G) JNK, but not of CREB, suggesting that PKCδ/JNK and CREB form part of two parallel and additive branches of the pathway. (H) Both rottlerin and the dominant-negative form of CREB (killer CREB, kCREB) blocked Ret- but not Pit-induced apoptosis. (I) Rottlerin blocked Ret-induced Pit-1 mRNA expression. (J) Schematic representation of the rat Pit-1 promoter, with known (Carneiro et al, 1998; Garcia et al, 2001) (inside boxes) and putative (outside boxes) response elements. Cells were cotransfected with Ret plus Pit-1 promoter-luciferase constructs with increasing deletions of the promoter. In the presence of Ret-S, Pit-1 promoter deleted to −2500 showed a three-fold induction in luciferase activity that was enhanced with further deletions, especially to −358, but that was lost with further deletion to −231. (K) The −358 bp region appears to be very important for the response to Ret, as the response was blocked by GDNF treatment and could not be induced by the Ret-SDN. (L) Both rottlerin and kCREB reduced Pit-1 promoter induction by Ret, although complete abolishment could only be achieved with both inhibitors together. (M) A consensus c/EBPα site was present in this small region of the promoter. Specific binding of c/EBPα to the Pit-1 promoter could be demonstrated by ChIP only 4 h after transfection with Ret. This was abolished by GDNF treatment and reduced by treatment with rottlerin or kCREB. c/EBPα Western blotting showed both the 42 and 30-kDa isoforms present in pituitary cell lines (Schaufele, 1996). Download figure Download PowerPoint The intracytoplasmic portion of Ret forms a complex with caspase 3 and PKCδ Our data suggested a strong relationship between the intracellular Ret portion, caspase 3, PKCδ, Pit-1 expression and apoptosis. It seemed that blockage of Ret or caspase-3 processing also inhibited Pit-1 expression. In fact, caspase-3 activity was detected as early as 2 h after transfection of either Ret-S, the kinase-dead mutant Ret-SKR or the intracellular portion of Ret (IC-Ret707–1017). However, there was no caspase-3 activity after transfection with Ret-SDN, a mutant that could not be processed by caspase-3 (Figure 5A, upper panel). GDNF was able to block Ret-induced caspase-3 activity, Pit-1 and apoptosis, but could not stop the apoptosis induced by IC-Ret707–1017, which correspondingly induced Pit-1 expression (Figure 5A, lower panels). We have found another caspase-3-processed protein, PKCδ (Voss et al, 2005), implicated in the pathway from Ret to the Pit-1 promoter. As both the ca
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