Laboratorio de Fisiología y Biología Molecular (D.K., D.R., A.C.L., D.H., O.A.C., E.A.), Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina; and Max Planck Institute of Psychiatry (M.P.P., G.K.S., F.H.), 80804 Munich, Germany
Address all correspondence and requests for reprints to: Dr. Eduardo Arzt, Laboratorio de Fisiología y Biología Molecular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Buenos Aires, Argentina. E-mail: earzt{at}bg.fcen.uba.ar.
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ABSTRACT |
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INTRODUCTION |
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Nur factors are the main mediators of CRH and cAMP stimulation of POMC in corticotrophs (14, 15, 16). Nur77, Nurr1, and NOR-1 are orphan nuclear receptors that belong to the Nur subfamily of transcription factors (17). They possess a highly conserved DNA binding domain that allows them to interact with the same DNA sequences in promoters, although specific and independent actions have also been described for these factors. Nur factors are particularly important in the regulation of the hypothalamic-pituitary-adrenal axis, acting on the hypothalamus (16), pituitary (14), and adrenal glands (18). In the pituitary, Nur77 and Nurr1 are involved in CRH-dependent induction of POMC mRNA, acting at two sites in the promoter: the NGFI-B response element (NBRE) site, which can bind one of these proteins as a monomer, and the Nur response element (NurRE) site, which can bind homodimeric Nur77/Nur77 or heterodimeric Nur77/Nurr1 proteins (14, 15, 19). The POMC NurRE (gTGATATTTacctccAAATGCCA) is an inverted copy of two NBRE (AAAGGTCA) sites with two mutations each, separated by 6 bp. The POMC NurRE is more sensitive to CRH stimulation than the NBRE site and may be responsible for stimulation of the POMC promoter under physiological conditions (14, 15, 19). NOR-1, the third member of the Nur subfamily, although capable of interacting with the consensus NurRE and NBRE sites, can only bind the POMC NurRE site and activate transcription as heterodimers together with Nur77 (15). The mechanisms and pathways that lead to Nur77/Nurr1 mRNA induction and transcriptional activation by CRH remain unknown. As CRH exerts its effect in corticotrophs through the type-1 CRH receptor (7), which raises intracellular cAMP levels, and Nur77/Nurr1 factors are involved in POMC induction by CRH, acting mainly at the NurRE site, in this paper we addressed the issue of calcium involvement in CRH and cAMP stimulation of NurRE-directed transcription.
The MAPK pathway is regulated by cAMP in several cell types. In view of this, we hypothesized they might play a role in CRH-Nur-POMC pathways. Rap1 is a small G protein that is activated by cAMP and PKA (20, 21). Depending on the cell type, Rap1 either inhibits or activates the MAPK pathway. Rap1 was shown to block MAPK activation through Ras (22, 23, 24), and the inhibition of MAPK activity exerted by Rap1 is thought to occur by interaction with Ras downstream effectors forming an inactive complex (25). In NIH3T3 and Rat-1 cells, the Raf-1 MAPK-kinase kinase activation by Ras is blocked by cAMP and PKA-dependent phosphorylation of Raf (26, 27). In addition, Rap1 activation by cAMP or a constitutively active form produced by point mutation of Rap1 inhibits Raf-1 activity (22). However, in several other cell types, activation of Rap1 by cAMP leads to activation of MAPK signaling (25, 28). The protein responsible for Rap1-dependent MAPK activation has been characterized in PC12 cells, in which cAMP stimulates MAPK signaling. In these cells, B-Raf is directly activated by Rap1 and activates the MAPK kinase (MEK), bypassing the negative effect of Rap1 on Raf-1 activation (28). There is a strong correlation between B-Raf expression and MAPK activation or inhibition in several cell types. Thus, for example, neurons express B-Raf, and in these cells cAMP stimulates MAPK activity; however, in astrocytes, in which B-Raf is not expressed, cAMP inhibits MAPK activity (29). Furthermore, constitutive overexpression of B-Raf in an astrocytoma cell line, in which cAMP inhibits MAPK, renders these cells sensitive to MAPK activation by cAMP (29). Thus, B-Raf expression is necessary for and confers cAMP-dependent stimulation of MAPK in several cell types, but has not been examined in AtT-20 corticotrophs.
In this work we analyze the transduction pathways involved in NurRE-dependent transcriptional activation by CRH and cAMP in AtT-20 corticotrophs. We demonstrate that the CRH/cAMP signaling that leads to Nur77/Nurr1 mRNA induction and transcriptional activation, and thus POMC expression, is dependent on PKA and involves calcium/calmodulin kinase II (CAMKII) (Nur induction/activation) and MAPK calcium-dependent and -independent (Nur phosphorylation-activation) pathways.
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RESULTS |
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We next examined whether this stimulation of NurRE-dependent transcriptional activity and calcium dependency is exerted at the level of Nur77 and Nurr1 mRNA expression. As shown in Fig. 2A, Nur77 and Nurr1 mRNA regulation correlate with NurRE-dependent transcriptional activity. CRH stimulation of Nur77 and Nurr1 mRNA was inhibited by NIF, and cAMP stimulation of Nur77 and Nurr1 mRNA was only partially inhibited (Fig. 2A
). Calcium entry by depolarization with KCl was sufficient to induce Nur77 mRNA but not Nurr1 mRNA (Fig. 2A
). The L-type calcium channel agonist FPL64176 increased the stimulation of Nur77 but did not modify Nurr1 expression (Fig. 2A
).
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To establish whether CRH, cAMP, and calcium regulation act at the Nur77 promoter, we transfected cells with a promoter Nur77-chloramphenicol acetyltransferase (CAT) construction. As expected from the above results, NIF treatment blocked CRH stimulation but only partially inhibited that of cAMP (Fig. 2B), indicating that calcium regulation is exerted at the transcriptional level.
Protein Kinase Dependence of CRH and cAMP Regulation of NurRE-Dependent Transcriptional Activity and Nur77/Nurr1 mRNA Expression
We next analyzed protein kinase involvement in CRH and cAMP actions by using pharmacological inhibitors of CAMKII: KN62; PKA: H89; and adenylate cyclase: MDL12330A. CRH stimulation of NurRE-dependent transcription was inhibited by all three compounds (Fig. 3A). cAMP stimulation of NurRE-dependent transcription was completely blocked by the PKA inhibitor but only partially inhibited by the CAMKII inhibitor (Fig. 3B
). The dependence on PKA was further confirmed by the inhibition of CRH or cAMP stimulation of Nur-RE by the expression of a dominant negative mutant of PKA (Fig. 3
, A and B). Simultaneous treatment with the CAMKII inhibitor and NIF produced the same partial degree of inhibition as either treatment on its own (Fig. 3B
). A 10-fold higher concentration of the CAMKII inhibitor produced the same partial inhibition (data not shown). The absence of synergistic or additive effects between these inhibitors suggests that they are blocking the same pathway, and that calcium exerts its effects through CAMKII. The existence of a calcium-independent pathway under cAMP stimulation is also confirmed. Concordant with the above mentioned effects on transcriptional activity, CRH induction of Nur77 is inhibited by the PKA and CAMKII inhibitors, and Nurr1 mRNA induction is inhibited by the CAMKII inhibitor (Fig. 3C
). cAMP induction of Nur77 and Nurr1 mRNA is inhibited by the PKA inhibitor and only partially blocked by the CAMKII inhibitor (Fig. 3C
).
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Characterization of the MAPK Cascade in AtT-20 Corticotrophs
To examine the activation of the MAPK transduction pathway by CRH and cAMP in corticotrophs, we studied their ability to activate Elk-1, a transcription factor activated by the ERK pathway. Cells were cotransfected with a Gal4 reporter plasmid and an expression vector for the chimeric Gal4 DNA binding domain fused to the transactivation Elk1 domain (Gal4-Elk1). CRH and cAMP increased Gal4-Elk1 activity (Fig. 5A); this increase was blocked by treatment with the MEK inhibitors PD98059 or UO126 or by expressing a dominant negative protein for MEK or Rap1b (RapN17) (Fig. 5A
), which has been shown to block Rap1 effects (28, 31). The constitutive active form of Rap1b (RapG12V) stimulated Elk1 transcription (Fig. 5A
). The presence of B-Raf has been shown to be necessary for MAPK activation by Rap1 in PC12 and other cell lines (28, 29, 32). We thus examined the expression of B-Raf mRNA in AtT-20 corticotrophs. We detected a single specific band by Northern blot, demonstrating constitutive expression of B-Raf (Fig. 5A
, inset). Overexpression of B-Raf protein enhanced the CRH stimulatory action on Gal4-Elk1 (Fig. 5A
).
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To establish whether activation of Rap1 by CRH and cAMP is involved in the activation cascade that stimulates NurRE, we analyzed CRH and cAMP stimulation of NurRE-dependent transcription in the presence of RapN17 and RapG12V. RapN17 expression inhibited NurRE activation by CRH and cAMP, whereas RapG12V potentiated CRH but not cAMP stimulation (Fig. 5C). A higher RapN17 concentration produced no greater inhibition of cAMP stimulation (data not shown). This result is in concordance with the lower activation of MAPK exerted by CRH with respect to cAMP (Fig. 5
, A and B); under a lower stimulation level of MAPK, RapG12V may increase the stimuli because the transduction pathway is not fully activated. RapN17 and RapG12V effects were highly specific insofar as: 1) these plasmids did not affect basal or IL-1-induced expression of kB-luciferase (LUC) reporter, a nuclear factor
B responding element (data not shown), ruling out nonspecific or toxic effects, and 2) overexpression of a dominant negative mutant of cAMP response element binding protein (CREB S133A) did not affect NurRE stimulation by CRH or cAMP (data not shown), confirming the specificity of the RapN17 action. These data indicate that Rap1 is involved in the MAPK pathway leading to NurRE activation in corticotrophs.
The PKA inhibitor completely blocked cAMP effects on Gal4-Elk1 transcriptional activation, but both NIF and the CAMKII inhibitor, as was the case for NurRE-LUC activity, only partially inhibited the stimulation by cAMP (Fig. 5D). NIF treatment or CAMKII or PKA inhibition almost completely blocked CRH stimulation (Fig. 5D
). These results show that activation of MAPK in AtT-20 cells is fully dependent on PKA activity, that calcium contributes to ERK activity and that cAMP activates the pathway also independently of calcium.
Direct MAPK Regulation of Nur77 Transactivation and Phosphorylation
Inhibition of MEK only slightly inhibited Nur77 and Nurr1 mRNA induction by cAMP and not by CRH (Fig. 6A). Thus, MAPK-dependent stimulation of NurRE by CRH is most probably exerted at a level other than that of Nur77 or Nurr1 expression. To determine whether stimulation of NurRE-dependent transcription by CRH, cAMP, and MAPK is exerted by direct regulation of Nur77 transactivation, we developed a chimeric Gal4-Nur77 construct and cotransfected cells with the Gal4 reporter plasmid. Both CRH and cAMP slightly but consistently increased Gal4-Nur77 basal transcriptional activity in AtT-20 cells (Fig. 6B
). Inhibition of MEK dramatically reduced basal transactivation activity of the Gal4-Nur77 protein and blocked CRH and cAMP stimulation (Fig. 6B
). The absence of action of the MEK inhibitor PD98059 on constitutive ß-galactosidase (ß-gal) expression (data not shown) and on CRH-induced Nur77/Nurr1 or Actin mRNA (Fig. 6A
) assures the specificity and absence of toxicity of the effect with this inhibitor. These results demonstrate that the regulation on NurRE activity by CRH and cAMP is controlled not only by induction of Nur77 and Nurr1 mRNAs but also by regulation of Nur77 transactivation and that the MAPK pathway is involved in both constitutive and CRH- or cAMP-stimulated transactivation activity of Nur77.
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Calcium, PKA, and MAPK Regulate the POMC Promoter
To establish whether NurRE-dependent transcriptional regulation by different protein kinases, including MAPK, also occurs in the context of the POMC promoter, we transfected AtT-20 cells with the POMC-LUC plasmid and tested the pharmacological inhibitors and dominant negative mutants on CRH and cAMP stimulation. Similar to their effect on NurRE-dependent transcription, the PKA and CAMKII inhibitors blocked POMC promoter activation by CRH, whereas the MEK inhibitors had only partial inhibitory effects (Fig. 7A). cAMP stimulation of the promoter was completely blunted by the PKA inhibitor and partially inhibited by the MEK and CAMKII inhibitors, although the latter had a much stronger effect than the former (Fig. 7B
). The expression of the dominant negative mutant for MEK or RapN17 inhibited both CRH and cAMP stimulation of the POMC promoter (Fig. 7
, A and B). Moreover, the constitutively active Rap1 (RapG12V) or MEK (MEK-EE) further increased the stimulation of POMC by Nur77, supporting the functional relevance of this pathway (Fig. 7B
). These results confirm that POMC regulation by PKA, CAMKII, and MAPK parallels that of Nur77/Nurr1 expression and transcriptional activation at the NurRE site.
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DISCUSSION |
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We demonstrate that Nur77 induction by CRH is dependent on calcium entry, and this effect is exerted at the level of the Nur77 promoter. The Nur77 promoter possesses no consensus cAMP response element sites that could be responsive to cAMP-PKA and calcium-CAMKII stimuli. The regulation of the Nur77 promoter in T cells by calcium involves two MEF2 sites located at positions -295 and -255 bp (35, 36, 37) and NFAT sites (37). Calcium-independent regulation also exists and involves different positions on the promoter. In contrast, the Nurr1 promoter contains consensus cAMP response element sites (38) that may confer both cAMP and calcium sensitivity.
The calcium-independent mechanism for Nur77/Nurr1 activation in AtT-20 cells can be shown with either CRH/NA or cAMP treatment. The fact that NA is necessary may be related to cAMP levels or PKA activation. cAMP levels are increased only 2-fold by CRH (39), and this level may not be sufficient for stimulation of POMC expression and Nur77/Nurr1 activation in the absence of calcium. Both calcium-dependent and -independent cAMP actions in AtT-20 cells have been demonstrated using specific protein phosphatases blockers (39). Furthermore, AtT-20 cells possess two isoforms of the PKA-regulatory subunits, type I and type II, the latter being the most abundant. Although cAMP treatment stimulates both isoforms, CRH only activates the type I isoform (9, 40). Thus, different PKA activation levels obtained by CRH, CRH/NA, or cAMP in AtT-20 cells may explain why the calcium-independent pathway is evidenced by cAMP or CRH/NA.
CRH- or cAMP-induced NurRE activation involves the MAPK signaling pathway, as demonstrated using dominant negative mutants of Rap1 or MEK, overexpression of MEK, or the MEK inhibitors PD98059 and UO126. Similarly, by using a Gal4-Elk1 transcription factor and ERK kinase assays, we show that MAPK activity is increased by CRH and cAMP treatments and depends on Rap1 activation. In addition, MAPK regulation of NurRE-directed transcription does not depend on induction of Nurr1 and Nur77 mRNA but on an increased transactivational activity of Nur77.
PKA-dependent and -independent activation of Rap1 by cAMP has been described in several models (21, 41, 42); our results show that in AtT-20 cells such activation is PKA dependent. B-Raf expression in AtT-20 cells is instrumental for cAMP-MAPK activation because it is the link that allows activation of MAPK by Rap1 (28, 32, 42). We show that the expression of B-Raf increases the CRH stimulation of the MAPK pathway. In several types of cells cAMP induces phosphorylation of Rap by PKA and inhibition of Raf activation by Ras (26, 27). B-Raf presence and activity allow the cells to bypass the inhibitory effect of Rap1 on Raf-1 and, consequently, on MAPK activity. B-Raf expression is cell type specific, and a correlation between stimulation of MAPK by cAMP and B-Raf presence can be drawn (32). In neurons that express B-Raf, cAMP treatment stimulates MAPK activity, whereas in astrocytes that lack B-Raf expression, cAMP treatment inhibits MAPK activity (29). Furthermore, constitutive expression of B-Raf in astrocytoma cells renders these cells sensitive to cAMP/Rap1-dependent stimulation of MAPK (29). Using the constitutive active form of Rap1 (RapG12V), we demonstrate that in AtT-20 cells the pathway linking Rap1 activation to ERK and increased Elk1 transcriptional activity exists and may be attributed to B-Raf presence, as was previously seen in PC12 cells (28). Furthermore, we show that RapG12V can activate basal Elk1 transcriptional activity and ERK activity, but does not stimulate the NurRE-LUC construct under basal conditions. This result is consistent with the fact that MAPK activity, in contrast to calcium and cAMP, does not increase Nur77 or Nurr1 expression levels, and in the absence of Nur77 protein no regulation by MAPK can be seen.
We show that in AtT-20 corticotrophs B-Raf is expressed, and using pharmacological (PD98059, UO126), biochemical (kinase and Nur77 phosphorylation assays), and molecular (Gal4-Elk1, RapN17, MEK dominant negative, and MEK expression vectors) tools, we demonstrate MAPK (Rap1/B-Raf/MEK/ERK1/2) induction by CRH and cAMP in those cells.
The studies with NIF show that downstream of PKA stimulation by CRH and cAMP, MAPK activation in AtT-20 cells involves not only calcium-dependent but also calcium-independent pathways, which may be particularly important for the calcium-independent stimulation of NurRE.
In addition to MAPK calcium-independent actions, it has been shown that in PC12 cells, MAPK activation by nerve growth factor is inhibited by an inhibitor of calmodulin, and that this effect is due to blockage of Raf-1 and B-Raf kinase activity (43). Calcium-dependent and -independent phosphorylation of CREB has also been described in PC12 cells; in this case, too, calcium-dependent phosphorylation requires the activation of the Rap1-B-Raf-MEK-ERK pathway (44). Thus, common regulatory pathways for MAPK regulation by calcium-dependent and -independent transduction signals seem to exist in corticotrophs and neuronal cells.
MAPK regulates Nur77 transactivation but not its induction. Both the Gal4-Nur77 basal and stimulated activities were blunted when cells were treated with the MEK inhibitor, indicating that both basal and stimulated activities of Nur77 are controlled by MAPK. Moreover, there is a direct regulatory action exerted by phosphorylation of Nur77 by ERK-2. In fact, Nur77 possesses two ERK phosphorylation sites at positions 108 and 325.
Basal MAPK activity may depend on autocrine and paracrine factors present in the culture medium. Overexpression of Nur77 without further stimulation is capable of increasing transcription at the NurRE and NBRE sites (14), indicating that the overexpressed protein is, at least in part, transcriptionally active. Basal MAPK activity is probably necessary for transcriptional activity of NurRE, since treatment with the MEK inhibitor severely decreases the transactivation of Gal4-Nur77. In NIH 3T3 fibroblasts, activation of CREB is dependent on basal MAPK activity. In these cells, although the MAPK pathway (Raf-MEK-ERK) is not stimulated by forskolin treatment, inhibition of the basal MAPK pathway by a dominant negative Raf protein or the pharmacological inhibitor of MEK (PD98059) can inhibit transcription of CREB without affecting Ser133 phosphorylation (45).
In conclusion, we show that CRH and cAMP trigger an array of calcium-dependent and -independent transduction signals, including a MAPK kinase pathway (Fig. 8). These different signals combine to regulate Nur77 and Nurr1 expression and activity, resulting in a unified control of POMC expression. As very recently stated, cAMP acts by different pathways including one through which PKA impacts the Rap-Raf-MAPK pathway in certain cell types and stages of differentiation (34). Furthermore, it is possible that in some cells both PKA-mediated phosphorylation of CREB and phosphorylation by other protein kinases (3-phosphoinositide-dependent kinase 1, protein kinase B, or MAPK) may be necessary for full activation of transcription (34). In this paper we demonstrate that AtT-20 corticotrophs constitute one important endocrine cell in which this pathway is relevant for control of POMC expression by Nur factors.
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MATERIALS AND METHODS |
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Cell Culture and Stimulation
AtT-20 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as previously described (46) under standard culture conditions in DMEM (Life Technologies, Inc.), with 10% fetal calf serum (FCS) supplemented with glutamine and antibiotics. Cells were treated as indicated in each experiment with human/rat CRH (100 nM ) (Bachem, Heidelberg, Germany), cAMP (500 µM ) (chlorophenylthio-cAMP), or KCl (36 mM ) plus the L-type calcium channel agonist FPL64176 (2 µM ) (RBI, Natick, MA). When used, NIF (1 µM ) and the inhibitors of protein kinases, MDL 12330 (10 µM ) (RBI), KN-62 (10 µM ) (RBI), H89 (10 µM ) (Calbiochem, San Diego, CA), UO126 (1020 µM ) (Calbiochem), and PD98059 (50 µM ) (Calbiochem) were added 15 min before other stimulatory treatment.
Plasmid Constructs
The plasmid constructs that we used were kindly provided and/or previously described: the NurRE-LUC, which contains three copies of NurRE sites coupled to the minimal POMC (-34/+63) promoter, and the Nur77 expression vector (pCMX-Nur77) were provided by Dr. Jacques Drouin (14, 19); the Nurr1 plasmid was obtained from Dr. Naganar Ohkura (47); the Nur77 promoter (-480) coupled to the CAT gene (Nur77promoter-CAT) came from Dr. Astar Winoto (35); the (770-bp fragment) POMC-LUC was obtained from Dr. Malcom Low (48); the Gal4-Elk1 expression vector and the B-Raf plasmid were provided by Dr. Phillip Stork (28); the expression vectors for RapN17 and RapG12V were supplied by Dr. Daniel Altschuler (49); the kB-LUC plasmid came from Dr. Michael Bell (50); the CREBS133A was provided by Dr. Beat Lutz (51); the MEK DN (dominant negative mutant of MEK) was supplied by Dr. Sivio Gutkind; the pBIND and pG5-LUC were obtained from Promega Corp. (Madison, WI); pMt-REV-PKA DN (dominant negative mutant of PKA) and pcDNA3 Ras N17, a dominant negative form of Ras and pcDNA3 MEK-EE, a constitutively active form of MEK, were described previously (52, 53). The Gal4-Nur77 plasmid was constructed as follows: the coding cDNA of pCMX-Nur77 plasmid was amplified with PCR using the CgCTCTAgAATgCCCTgTaTCCAAgCTC and ggggTACCTCAgAAAgACAAggTg primers. The fragment containing the coding Nur77 sequence was cloned using the NcoI (blunt) and KpnI enzymes in-frame to the pBIND vector previously cut with EcoRV and KpnI. Correct in-frame insertion was verified by sequencing.
Northern Blot
For Northern blot analysis, cells were plated on six-well plates, at a density of 400,000 cells per well in DMEM 10% FCS supplemented. After overnight attachment of cells, medium was changed for DMEM without serum and left overnight. After overnight washout, cells were treated with different stimuli. At the indicated times total RNA was isolated by the guanidine isothiocyanate-phenol-chloroform method. RNA extraction, denaturing gel electrophoresis, Northern blotting, preparation of radiolabeled probes, and hybridization procedures were performed as described previously (46). Briefly, 5- to 10-µg RNA samples, as measured at 260 nM, were denatured with glyoxal, electrophoresed on a 1.2% agarose gel, and transferred overnight to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The filters were baked for 2 h at 80 C, prehybridized for 4 h at 60 C, and then hybridized in the same solution (50% formamide, 5x sodium chloride/sodium phosphate/ EDTA, 5x Denhardts solution, 0.1% sodium dodecyl sulfate, 100 µg/ml denatured salmon sperm DNA) with the addition of the radiolabeled probe at the same temperature for 12 h. Each blot was washed under increasingly stringent conditions of salt and temperature with a final wash of 30 min at 60 C in 0.1x sodium chloride/sodium citrate containing 1% sodium dodecyl sulfate. Dried filters were exposed to Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) at -70 C with intensifying screens for 6 h to 4 d.
The following probes were used: a fragment of 1.4 kb (PvuII digest) from Nur77 cDNA (14, 19); the B-Raf cDNA (640 bp) (28), a fragment of 500 bp of Nurr1 cDNA (HindIII, PstI digest) (47), and a 1-kb Pst-1 digest cDNA for actin (54). The cDNA probes were labeled with a random-primed kit with -32P-dCTP (Amersham Pharmacia Biotech). The autoradiograms were scanned with an ultroscan II laser densitometer (LKB, Rockville, MD). The blots were reprobed after eluting the first radiolabeled probe by washing according to the manufacturers instructions in 5 mM Tris/HCl (pH 8.0), 2 mM EDTA, 0.1x Denhardts solution, at 65 C for 2 h. After the previous signal was removed, confirmed by reexposure of the autoradiogram, the blots were prehybridized and hybridized following the methods described above.
Transient Transfection and Reporter Luciferase, CAT, and ß-gal Assays
Cell transfection was performed with lipofectamine using standard procedures, as previously described (55). After plating the cells in six-well plates, the cells were transfected for 6 h in OPTIMEM (Life Technologies, Inc.) using 8 µl of lipofectamine (Life Technologies, Inc.) per well, with the following plasmids, as indicated: NurRE-LUC (500 ng), POMC-LUC (500 ng), Nur77promoter-CAT (500 ng), Gal4-Elk1 (300 ng) or Gal4-Nur77 (300 ng) plus the reporter for Gal4 pG5-LUC (500 ng), RapN17, RapG12V, PKA DN, MEK DN, RasN17, B-Raf expression vectors (300 ng), and MEK-EE (30 ng), in conjunction with a control rous sarcoma virus (RSV)-ß-gal construction (200 ng). Cells were then washed and left overnight in DMEM without serum. The next day cells were washed and stimulated for 6 h in serum-free DMEM with the indicated stimuli. After treatment, LUC activity in cell lysates was measured as previously described (55) using the Luciferase measure kit (Promega Corp.) with a Junior luminometer (Berlthold, Bad Wildbad, Germany). CAT and ß-gal activities were measured as previously described (55), the last as control for transfection efficiency. Results were standardized for ß-gal activity. Empty vectors for POMC-LUC and NurRE-LUC constructs showed no stimulation by CRH or cAMP (data not shown).
MAPK Assay, Nur77 Phosphorylation, and Western Blot
AtT-20 cells were cultured in six-well plates in DMEM 10% FCS supplemented with glutamine and antibiotics; 24 h later cells were washed out and left for another 24 h in serum-free DMEM. Stimulation was performed for 2, 5, or 10 min with CRH, cAMP, or KCl plus the L-type calcium channel agonist FPL64176. Cellular extracts were collected using a kinase lysis buffer (HEPES, 20 mM, pH 7.5; EGTA, 10 mM; ß-glycerolphosphate, 40 mM; NP-40, 1%; MgCl2, 2.5 mM; orthovanadate, 2 mM; phenylmethylsulfonyl fluoride, 1 mM; dithiothreitol, 1 mM; leupeptin, 20 µg/ml). After centrifugation, supernatants were split to detect total (phosphorylated and unphosphorylated) ERK2 protein and phosphorylated ERK1/2 protein contents by Western blot as described previously (53). Briefly, after separation of proteins by SDS-PAGE (10% polyacrylamide) and blotting onto nitrocellulose membranes, immunoblot analysis was performed with antiphospho-ERK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After stripping, the membranes were treated with the anti-ERK2 antibody (Santa Cruz Biotechnology, Inc.). Detection was performed by chemiluminescence (Amersham Pharmacia Biotech) for antiphospho-ERK antibody and with the phosphatase alkaline method (Life Technologies, Inc.) for the anti-ERK2 antibody. Extracts were used for immunoprecipitation and kinase assay as described previously (56). Briefly, the supernatants were immunoprecipitated with anti-ERK2 (Santa Cruz Biotechnology, Inc.) antibody. After incubation at 0 C for 90 min, protein A/G plus-Agarose beads (Santa Cruz Biotechnology, Inc.) were added and left in agitation at 4 C for 30 min. After precipitation the pellet was washed three times with 500 µl of wash buffer 1 (PBS; NP-40 1%; vanadate, 2 mM ), one time with 500 ml of wash buffer 2 (Tris, 100 mM, pH 7.5; LiCl, 0.5 M ) and one time with the kinase reaction buffer [3-(N-morpholino)propane sulfonic acid, 4.2 mM; ß-glycerophosphate, 4.2 mM; MgCl2, 2.5 mM; EGTA 0.2 mM; sodium fluoride, 0.2 mM; vanadate, 0.2 mM ).
Each pellet was incubated with 30 µl of the reaction buffer plus 32ATP (10 µCi), myelin basic protein (MBP) (10 µg), dithiothreitol (3 mM ), and rATP at 37 C for 30 min. The cracking buffer (10 µl) was added and the products boiled for 5 min to denaturate the MBP. After electrophoresis, the gel was dried for 2 h at 80 C and exposed to an autoradiographic Kodak XAR5 film at -70 C with intensifying screens for 6 h to 4 d. For phosphorylation of Nur77 protein the assay was performed in vitro as follows: AtT-20 cells were transiently transfected with a 500 ng of the Gal4-Nur77 construction and, after 44 h, cells were treated with 50 µM PD98059 for 4 h. Cellular extracts were obtained and the protein was immunoprecipitated using polyclonal goat IgG anti the C-terminal part of Nur77 antibody (Santa Cruz Biotechnology, Inc.), as described above. The immunoprecipitated protein was used as in vitro substrate for the kinase reaction (performed as described above) with mouse ERK2 immunoprecipitated from AtT-20 cells treated under different conditions. Western blot analysis for Gal4-Nur77 was performed using the polyclonal IgG anti-Nur77 antibody already described and a second antigoat coupled to phosphatase alkaline antibody (Santa Cruz Biotechnology, Inc.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Members of the Argentine National Research Council (CONICET).
Abbreviations: CAMKII, Calmodulin kinase II; CAT, chloramphenicol acetyl transferase; CREB, cAMP response element binding protein; FCS, fetal calf serum; ß-gal, ß-galactosidase; LUC, luciferase; MBP, myelin basic protein; MEK, MAPK kinase; MEK DN, dominant negative mutant of MEK; MEK-EE, MEK constitutively active form expression vector; NA, noradrenaline; NBRE, NGFI-B response element; NIF, nifedipine; NurRE, Nur response element; POMC, proopiomelanocortin; PKA, protein kinase A; PKA DN, dominant negative mutant of PKA; RSV, rous sarcoma virus.
Received for publication August 16, 2001. Accepted for publication February 26, 2002.
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REFERENCES |
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