TY - JOUR
AU - Zakin, Mario, M.
AB - Studies in Nur77-deficient mice have shown that the basal regulation of hypothalamic and pituitary functions as well as the adrenocortical steroidogenesis in these animals is normal. This indicates that Nur77-related orphan receptors may substitute Nur77 functions in the hypothalamo-pituitary-adrenal axis by a compensatory mechanism. Nor1 is the most recently cloned member of the NGFI-B/Nur77 subfamily, and its properties are still largely unknown. We demonstrate here that Nor1 is expressed in the pituitary gland and adrenal cortex, and that ACTH and angiotensin II (AngII) treatment of adrenal fasciculata cells induces Nor1 expression. Time-course analysis with both hormones on steroidogenic capacity and the specific gene expression in adrenal cells strongly suggest that Nor1 is an intermediate in the long-term consequences of ACTH or AngII treatment. The Nor1 and NGFI-B/Nur77 amino acid sequence homology and the analysis of the trans-activation properties of Nor1 show that the overall structural and functional organization of the two proteins is similar. As observed with NGFI-B/Nur77, Nor1 activates the expression of genes encoding steroidogenic enzymes as P450c21, through its interaction with NGFI-B response element promoter sequences. In contrast, binding experiments of Nor1 with the palindromic NurRE sequence suggest that Nor1 is not an efficient substitute for the NGFI-B/Nur77 activation of the POMC gene expression in pituitary glands. All these results indicate that Nor1 and NGFI-B/Nur77 may play similar albeit distinct roles in the hypothalamo-pituitary-adrenal axis. Further experiments also show that the mechanisms responsible for the transcriptional regulation of Nor1 in adrenal cells appear to depend on the protein kinase A and protein kinase C cascades. NOR1 IS A member of a related subgroup of proteins belonging to the nuclear receptor superfamily (1). This subgroup includes Nurr1 (also known as RNR1, NotI, HZF-3) (2, 3) and NGFI-B (also known as Nur77, NAK1, N10) (4, 5). These three proteins share the greatest similarity in their DNA-binding domains (91–97% sequence homology), and also contain large regions of homology in the carboxyl region, but diverge significantly in their amino terminal sequences. Nor1, Nurr1 and NGFI-B/Nur77 (for simplicity Nur77) when acting as monomers, bind to the same NGFI-B response element, NBRE (6). In contrast with the other two proteins, Nor1 is unable to promote ligand-induced activation of the retinoid X receptor (RXR) bound to its specific DNA response element, because of its inability to form heterodimers with RXR (7, 8). Recently, a novel mechanism for Nur77 activity has been proposed that involves the formation of homodimers. These dimers interact with NurRE, a palindromic response element containing a six-nucleotide spacing between its half-sites. Moreover, both halves of the palindromic NurRE are required for responsiveness to physiological signals (9). Previous work comparing the spatio- temporal distribution of Nor1, Nurr1, and Nur77 messenger RNAs (mRNAs) showed complex patterns of expression among the three receptors, with both distinct and overlapping patterns, and predominant expression in the central nervous system (8). CRH, the main physiological regulator of ACTH secretion and synthesis, is synthesized in the hypothalamus by the parvicellular neurons. These cells secrete CRH, which is delivered to the anterior pituitary gland, and through the cAMP pathway stimulates the secretion of the preformed ACTH. The CRH-induced increase in cAMP also stimulates the expression of the POMC gene and synthesis of a 30-kDa prohormone. Proteolytic cleavage of this protein leads to ACTH and β-lipotropin production. ACTH, together with the octapeptide angiotensin II (AngII), control adrenal steroidogenesis in vivo. ACTH is the physiological regulator of the synthesis and secretion of glucocorticoids by the adrenal cortex, whereas AngII regulates the synthesis and secretion of mineralocorticoids. However, ACTH is also able to stimulate mineralocorticoid secretion, and AngII can stimulate glucocorticoid secretion as well. The parvicellular neurons, together with the pituitary corticotroph cells and adrenals, constitute the hypothalamo-pituitary-adrenal (HPA) axis that mediates the stress response. The expression of Nur77 in the cells of this system suggests that the Nur77 protein performs signaling functions at all levels of the HPA axis. Indeed, Nur77 transcription has been shown to be induced in CRH neurons (10). In pituitary-derived cells, CRH-induced Nur77 activates transcription of the POMC gene through formation of protein homodimers that interact with the NurRE sequence present in the POMC promoter region (9). In adrenal glands, in vitro and ex vivo experiments have shown that this nuclear receptor acts as a mediator of ACTH-induced gene expression, resulting in increased steroidogenesis. The promoter sequences required for the synthesis of ACTH-regulated steroidogenic enzymes, such as the 21-hydroxylase (P450c21), contain NBRE-binding sites that are recognized and activated by Nur77 (11). However, studies of adrenocortical function and regulation of the steroid P450c21 gene in the Nur77-deficient mouse demonstrated that the adrenal gland functions normally in these animals, indicating that Nur77 is not essential for HPA axis function (12). This result suggests that other members of the same subfamily of nuclear receptors are sufficient to maintain normal steroidogenesis in vivo by a compensatory mechanism. Nor1 is the latest cloned member of the NGFI-B/Nur77 subgroup (1), and most of its properties are still unknown. In this report, the trans-activation and DNA-binding properties of the Nor1 protein, and the physiological effectors involved in its gene regulation were examined. The results are compared with those obtained for NGFI-B and indicate that the two proteins may play similar, albeit distinct, roles in adrenal cell function. Materials and Methods Plasmids pSG5-Nor1 was constructed by cloning a 2.9-kb XbaI-EcoRI fragment of Nor1 complementary DNA (cDNA) (1) into pSG5 (Stratagene, La Jolla, CA). pSG5-Nur77 was generated by cloning a 2.48-kb EcoRI fragment of the pJDM3 construct (4) into pSG5. pΔN are constructs in which nucleotides from the 5′-end of Nor1 cDNA were deleted, and pC are constructs in which nucleotides from the 3′-end were deleted. The resulting plasmids included the following deletions in the corresponding products: pΔN7–32, amino acid residues 7–32; pΔ7–118, amino acid residues 7–11; pΔN7–210, amino acid residues 7–210; pC1–408, amino acid residues 409–628; and pC1–532, amino acid residues 533–628. Some of these deletion mutants were generated by PCR and ligation of amplified fragments using pSG5-Nor1 as a template. pΔN7–32, pΔN7–118, and pΔN7–210 were generated using oligonucleotides 5′–CCCGACTATGCCAAGCTG, 5′-ATTCCTCCTCCCTCTGGCC, and (5′-CACCTGGGCTATGACCCC as forward primers, respectively, and oligonucleotide 5′-GGCTTGCACGCAGGGCAT as the reverse primer. To generate the plasmid pC1–532, pSG5-Nor1 was digested with EcoRV and BglII; the BglII site was then blunt ended, and the DNA was religated. pC1–408 was obtained using the Gene-Editor In Vitro Site-Directed Mutagenesis System (Promega Corp., Madison, WI), by introducing a stop codon into the triplet encoding Leu408. The oligonucleotide (5′-AGATCTTGCGTCTGTTTAAGCTCGGAC) was used to introduce this latter mutation. The chloramphenicol acetyltransferase (CAT) reporter plasmid, p3NBRE-TK-CAT, contains three copies of the NBRE site cloned into pBLCAT5 upstream of the herpes simplex virus thymidine kinase promoter (13). The p−72/65 P450c21-TK-CAT contains two copies of the −65 element of the P450c21 5′-region cloned into pBLCAT5 upstream of the thymidine kinase promoter. The p5′-P450c21-luc or CAT contains the −150/+9 region of cytochrome P450c21 cloned into the pGL3-basic vector or into the pCAT3-basic vector, respectively (Promega Corp.). RNA extraction and Northern blots Total cellular RNA was isolated by the guanidinium thiocyanate procedure (14). RNA integrity was verified by denaturing formaldehyde agarose gel electrophoresis and quantified by measuring absorbance at 260 nm. Total RNA (24 μg) was first denatured in 2.2 m formaldehyde and 50% formamide at 55 C for 15 min and then run on a 1.5% formaldehyde agarose gel. RNA was blotted onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Aylesbury, UK) by capillary transfer. The membranes were hybridized with 32P-labeled Nor1 or NGFI-B probes at 42 C in a buffer containing 50% formamide, 5 × SSC (standard saline citrate), 4 × Denhardt’s solution, 0.1% SDS, and 10 μg/ml salmon sperm DNA. A β-actin probe was used to normalize RNA loading between lanes. The Nor1 probe was a 409-bp fragment containing the sequences between nucleotides 1908–2317. This fragment was obtained by PCR, using rat Nor1 cDNA as a template. In situ hybridization The method used was essentially that previously described (15). Oligodeoxynucleotides complementary to nucleotides 1191–1238 and 1272–1319 of the Nor1 sequence were 3′-end labeled to a specific activity of 200–600 kBq/pmol, using[ 33P]deoxy-ATP (NEN Life Science Products, Boston, MA) and terminal deoxynucleotidyl transferase (Roche, Indianapolis, IN) following the manufacturer’s specifications. Frozen adrenal glands from adult Sprague Dawley rats (250–350 g) were cryostat sectioned (14 μm), thaw-mounted on poly-l-lysine-coated slides, and stored at −80 C for 1–3 days. Experiments were performed according to the methods of Young et al. (16), with the modifications described by Zoli et al. (17). Probes were added to a concentration of 0.55 nm (corresponding to ∼15 fmol/section). Controls with nonradioactive oligonucleotides were performed as previously described (17). Cell culture COS cells were grown in DMEM containing 4.5 g/liter glucose supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100μ g/ml), fungizone and amphotericin (2.5 μg/ml), l-glutamine (2 mm), and sodium pyruvate (10 mm) at 5% CO2 in 95% air. Y1 cells were grown in DMEM/F-12 nutrient mix (1:1) supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), fungizone and amphotericin (2.5 μg/ml), and l-glutamine (2 mm) at 5% CO2 in 95% air. Cells were stimulated with forskolin (2 × 10−6m; Sigma), 12-O-tetradecanoylphorbol 13-acetate (TPA; 2 × 10−7m; Sigma) or ACTH (10−8m; Sigma) for various durations as indicated below. Bovine fasciculata adrenal cells were prepared by sequential treatment of adrenal cortical slices with trypsin (0.15%) and cultured as previously described (18). Cells were stimulated with ACTH (10−8m), AngII (10−7m), forskolin (2 × 10−6m), or TPA (2 × 10−7m) for various durations. The human epithelial carcinoma C33 cells were cultured as previously described (19). Transient expression assays Twenty-four hours before transfection, 5 × 105 cells were plated in 3-cm dishes, then the culture medium was changed 3 h before transfection. Plasmids (prepared with the QIAGEN plasmid kit (QIAGEN, Hilden, Germany) were introduced into cells using the FuGENE 6 Transfection Reagent (Roche) according to the manufacturer’s instructions or by the calcium phosphate precipitation technique (19). Each dish was transfected with 5 fmol p3NBRE-TK-CAT, p2NBREc21-TK-CAT, or p5′-450c21-CAT; 50 fmol pRSV-β-galactosidase; 25 fmol expression vector (pSG5-Nor1 or pSG5-Nur77 or the pSG5-Nor1 mutants); or control plasmid (pSG5); and 25 fmol carrier plasmid DNA (pBluescript KS+, Stratagene, La Jolla, CA), which corresponded to about 2.0 μg/dish. Seventy-two hours posttransfection, cells were washed, collected by centrifugation, resuspended in 250 mm Tris-HCl and 15% glycerol (pH 7.5), and lysed by four freeze-thaw cycles. Protein concentrations were determined by the Bradford assay, and CAT activity was measured by incubating 20 μg protein with 0.2 μCi[ 14C]chloramphenicol (20 μCi/mmol) and 250μ m butyryl-coenzyme A in 100 ml 250 mm Tris-HCl, pH 7.5, for 1.5 h at 37 C. Acylated chloramphenicol was extracted using a mixture of 200 μl 2,6,10,14-tetramethylpentadecane and xylene (2:1) and counted in a scintillation counter (20). The relative CAT activities were normalized to β-galactosidase activity. Gel mobility shift assays Canonical NBRE (5′-GATCGAGTTTTAAAAGGTCATGCTCAATTT-3′), −72/−65 P450c21 (5′-GCTCTAGAAGCAAAGGTCAGAGCTC-TAGAGC-3′), NurRE (5′-TCCTAGTGATATTTACCTCCAAATGCCAGGA-3′) and MATE (5′-ATTAGAAAAGAGGAAGGAAATT-3′) oligonucleotides were 5′-end labeled with T4 polynucleotide kinase and purified on MicroSpin G-25 columns (Pharmacia Biotech). The oligonucleotide MATE contains a binding site for the transcription factor PU.1, present in the promoter of the receptor Fcγ gene. The DNA binding motifs are underlined. Cell protein extracts and DNA-protein mobility shift assays were performed as previously described (21, 22). The anti-NGFI-B 2E1 monoclonal antibody was used in the supershifting experiments (11). In this case, the antibody was preincubated with the protein extracts for 10 min at room temperature before probe addition. Results Nor1 is expressed in the adrenal cortex and pituitary gland Northern blot analysis of rat tissues showed that Nor1 is expressed in the adrenal and pituitary glands (Fig. 1a). As in fetal and adult brain (1, 23), a predominant 6.5-kb transcript was observed, as well as a second 5- to 5.5-kb transcript. In situ hybridization experiments showed that the nuclear receptor was expressed in the adult mesoderm-derived adrenal cortex (Fig. 1b), suggesting a role for Nor-1 in adrenocortical function. This result is consistent with mRNA expression previously observed in the adrenal cortex of embryonic day 16.5 rat embryos (8). Figure 1. Open in new tabDownload slide Nor1 is expressed in the pituitary and adrenal gland cortex. a, Northern blot analysis of RNA species in the rat. Pi, Pituitary gland; Hi, hippocampus; Th, thalamus; A, adrenal glands; Ce, cerebellum; Co, cortex. Considering the expression in adrenal glands as 100%, with the signals being normalized to those of β-actin controls, expression in the pituitary gland was 12%, that in the hippocampus was 26%, that in the thalamus was 8%, that in the cerebellum was 4%, and that in the cortex was 33%. b, Brightfield photographs of film autoradiographs showing distribution of Nor1 mRNAs in the cortex of rat adrenal glands (A, B, and C correspond to three separate experiments). D, An excess of the unlabeled oligonucleotide probe was added as a control for specificity. Figure 1. Open in new tabDownload slide Nor1 is expressed in the pituitary and adrenal gland cortex. a, Northern blot analysis of RNA species in the rat. Pi, Pituitary gland; Hi, hippocampus; Th, thalamus; A, adrenal glands; Ce, cerebellum; Co, cortex. Considering the expression in adrenal glands as 100%, with the signals being normalized to those of β-actin controls, expression in the pituitary gland was 12%, that in the hippocampus was 26%, that in the thalamus was 8%, that in the cerebellum was 4%, and that in the cortex was 33%. b, Brightfield photographs of film autoradiographs showing distribution of Nor1 mRNAs in the cortex of rat adrenal glands (A, B, and C correspond to three separate experiments). D, An excess of the unlabeled oligonucleotide probe was added as a control for specificity. ACTH and AngII induce the expression of Nor1 mRNA in primary cultures of fasciculata adrenal cells In the adrenal cortex, ACTH has a rapid stimulatory effect on steroidogenesis by up-regulating the synthesis of adrenal steroids. Primary cultures of adrenal fasciculata cells serve as a well defined steroidogenic model (24, 25). When these cells were treated with ACTH (10−8m) for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, the synthesis of cortisol was rapidly stimulated and was still increasing after 8 h of treatment (Fig. 2a). Nor1 mRNA synthesis was also rapidly induced, with an induction peak being reached within 2 h of treatment. Expression of the Nor1 gene clearly precedes that of the steroidogenic enzymes, such as cytochrome P450c17 and 3β-hydroxysteroid dehydrogenase (Fig. 2a). In addition, the expression of P450c21 was significantly induced after 24 h of hormonal treatment (data not shown). AngII is another hormone that controls in vivo adrenal steroidogenesis, and its effect on the steroidogenic responsiveness of cultured bovine adrenal fasciculata cells is well documented (24). When these cells were treated with AngII (10−7m), cortisol synthesis was rapidly stimulated, although to a lower extent than with ACTH treatment. AngII treatment also induced Nor1 mRNA synthesis, but in this case transcription of Nor1 began later than in the case of ACTH, and P450c17 expression followed immediately this transcription (Fig. 2b). These results clearly show that both hormones induce Nor1 expression in primary cultures of fasciculata cells and also indicate that the Nor1 nuclear receptor may function as an intermediate in the long-term effects of these hormones on adrenal steroidogenesis and cell differentiation. Figure 2. Open in new tabDownload slide ACTH and AngII treatment of fasciculata adrenal cells increases Nor1 mRNA levels. Northern blot analysis of Nor1, P450c17, 3β-hydroxysteroid dehydrogenase, β-actin, and 28S RNAs obtained from cells treated with ACTH (10−8m; a) and AngII (10−7m; b). a) Lanes 0 and 8, Untreated cells; lanes 1–7, fasciculata cells treated with ACTH for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively; lanes 9–12, fasciculata cells treated with ACTH for 1.5, 3, 6, and 12 h, respectively. The synthesis of cortisol was measured in the culture medium. b) Lanes 13–17, Fasciculata cells treated with AngII for 45 min, 1.5 h, 2.5 h, 4 h, and 8 h, respectively; lanes 18–21, fasciculata cells treated with AngII for 1.5, 3, 6, and 12 h, respectively. The synthesis of cortisol was measured in the medium. In untreated adrenal cells, the production of cortisol was 4.3 ng/106 cells at 2 h and 8.5 ng/106 cells at 8 h. Figure 2. Open in new tabDownload slide ACTH and AngII treatment of fasciculata adrenal cells increases Nor1 mRNA levels. Northern blot analysis of Nor1, P450c17, 3β-hydroxysteroid dehydrogenase, β-actin, and 28S RNAs obtained from cells treated with ACTH (10−8m; a) and AngII (10−7m; b). a) Lanes 0 and 8, Untreated cells; lanes 1–7, fasciculata cells treated with ACTH for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively; lanes 9–12, fasciculata cells treated with ACTH for 1.5, 3, 6, and 12 h, respectively. The synthesis of cortisol was measured in the culture medium. b) Lanes 13–17, Fasciculata cells treated with AngII for 45 min, 1.5 h, 2.5 h, 4 h, and 8 h, respectively; lanes 18–21, fasciculata cells treated with AngII for 1.5, 3, 6, and 12 h, respectively. The synthesis of cortisol was measured in the medium. In untreated adrenal cells, the production of cortisol was 4.3 ng/106 cells at 2 h and 8.5 ng/106 cells at 8 h. Nor1 amino- and carboxyl-terminus are important for transcriptional activation By sequence homology, the nuclear receptor, Nor1, shares a common organization of structural domains with other members of the NGFI-B/Nur77 subfamily. To gain further insight into the function of each of its structural domains, a series of Nor1 mutants was constructed. These constructs were cotransfected with a 3NBRE-TK-CAT reporter plasmid in the Y1 cell line, as NBRE was previously reported to be a specific DNA-binding site for the protein (1, 23). The data in Fig. 3a indicate that Nor1 contains at least two distinct regions, which when coupled to its DNA-binding domain, promote transcription from NBRE sites. These regions correspond to the two main trans-activation domains, AF-1 and AF-2, previously described in several nuclear receptors including Nur77 (26). In Nor1, AF-1 is localized between amino acid residues 1–118, at the amino-terminus of the protein, whereas AF-2 is located between amino acid residues 532–628, at the carboxyl-terminus. These results and a comparison of the amino acid sequences indicate that the overall structural and functional organization of Nor1 is similar to that of Nur77. Figure 3. Open in new tabDownload slide Transcriptional activity of wild-type Nor1 and amino- and carboxyl-terminal mutants. a, Y1 cells were transfected with p3NBRE-TK-CAT, pSG5, pSG5-Nor1, or the pSG5-Nor1 mutants along with the pRSV-β-galactosidase vector. CAT activities were normalized by measuring relative β-galactosidase activity for each construct. The results presented are the average of three experiments using two or three different plasmid DNA preparations. As a control for the expression of Nor1 and its mutants, total protein extracts from each transfectant were analyzed for specific interaction with a NBRE oligonucleotide in gel mobility shift assays; with the exception of pC1–532, all extracts contained a significant amount of expressed protein (data not shown). b, Schematic illustration of the structural organization of a nuclear receptor; the evolutionary conserved regions C (DNA-binding domain) and E (putative ligand-binding domain) and divergent regions A/B, D, and F are indicated; region F is absent in the NGFI-B/Nur77 subfamily. c, Nor1 is similarly represented. The DNA-binding domain located between amino acid residues 293 and 379 is represented by a hatched box, and the positions of the mutations are indicated. Figure 3. Open in new tabDownload slide Transcriptional activity of wild-type Nor1 and amino- and carboxyl-terminal mutants. a, Y1 cells were transfected with p3NBRE-TK-CAT, pSG5, pSG5-Nor1, or the pSG5-Nor1 mutants along with the pRSV-β-galactosidase vector. CAT activities were normalized by measuring relative β-galactosidase activity for each construct. The results presented are the average of three experiments using two or three different plasmid DNA preparations. As a control for the expression of Nor1 and its mutants, total protein extracts from each transfectant were analyzed for specific interaction with a NBRE oligonucleotide in gel mobility shift assays; with the exception of pC1–532, all extracts contained a significant amount of expressed protein (data not shown). b, Schematic illustration of the structural organization of a nuclear receptor; the evolutionary conserved regions C (DNA-binding domain) and E (putative ligand-binding domain) and divergent regions A/B, D, and F are indicated; region F is absent in the NGFI-B/Nur77 subfamily. c, Nor1 is similarly represented. The DNA-binding domain located between amino acid residues 293 and 379 is represented by a hatched box, and the positions of the mutations are indicated. Nor1 interacts as a monomer with NBRE sequences, but fails to form homodimers capable to interact with the palindromic NurRE motif of the POMC promoter Within the nuclear receptor family, Nur77 is distinguished by its ability to bind the NBRE site as a monomer (27). NBRE contains a single half-site of the estrogen receptor class composed of the hexanucleotide AGGTCA with two A residues preceding the hexanucleotide (6). To examine the DNA-binding properties of Nor1, protein extracts of COS cells transfected with a Nor1 expression vector were analyzed by gel retardation assays. We first studied the interaction of these extracts with a labeled oligonucleotide containing the canonical NBRE site (Fig. 4a). Two protein-DNA complexes were observed that were specifically competed by an excess of cold NBRE oligonucleotide, but not by a heterologous oligonucleotide. Similar protein-DNA complexes were detected when nuclear extracts from adrenal glands were used (Fig. 4b); in the latter case, a faster migrating complex was also observed. As the band corresponding to this complex was partially supershifted when antibodies specifically directed to Nur77 were used, we concluded that at least part of this complex corresponds to Nur77 bound to NBRE. Figure 4. Open in new tabDownload slide Gel mobility shift assays demonstrate that Nor1 interacts with the NBRE site, but not with the palindromic NurRE sequence. a, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 0, Protein extracts from untransfected COS cells in the presence of NBRE; lane 1, binding between extracts from pSG5-Nor1-transfected COS cells and NBRE; lanes 2 and 3, as in lane 1, with the addition of a 10- or 25-fold excess of unlabeled NBRE, respectively; lane 4, as in lane 1, with the addition of a 25-fold excess of an unlabeled heterologous oligonucleotide MATE. b, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from mouse adrenal glands. Lane 1, Binding between protein extracts from pSG5-Nor1-transfected COS cells in the presence of NBRE; lane 5, binding between protein extracts from mouse adrenal glands and NBRE; lane 6, as in lane 5, with the addition of antibodies directed against Nur77; lanes 7 and 8, as in lane 5, with the addition of a 10- or 25-fold excess of unlabeled NBRE, respectively. c, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 1, As in a and b. Lane 9, Protein extracts from untransfected COS cells in the presence of NurRE; lane 10, binding between extracts from pSG5-Nor1 transfected COS cells and NurRE; lane 11, binding between extracts from pSG5-Nur77 transfected COS cells and NurRE; lane 12, as in lane 11, with the addition of a 30-fold excess of unlabeled NurRE. The short arrows indicate the specific monomeric NBRE-Nor1 complexes; the long arrows indicate the specific monomeric NBRE-Nur77 or NurRE-Nur77 complexes. The open long arrow indicates the specific dimeric NurRE-Nur77 complex. The arrowhead indicates the mobility of the supershifted complex obtained in the presence of antibodies directed against Nur77. Figure 4. Open in new tabDownload slide Gel mobility shift assays demonstrate that Nor1 interacts with the NBRE site, but not with the palindromic NurRE sequence. a, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 0, Protein extracts from untransfected COS cells in the presence of NBRE; lane 1, binding between extracts from pSG5-Nor1-transfected COS cells and NBRE; lanes 2 and 3, as in lane 1, with the addition of a 10- or 25-fold excess of unlabeled NBRE, respectively; lane 4, as in lane 1, with the addition of a 25-fold excess of an unlabeled heterologous oligonucleotide MATE. b, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from mouse adrenal glands. Lane 1, Binding between protein extracts from pSG5-Nor1-transfected COS cells in the presence of NBRE; lane 5, binding between protein extracts from mouse adrenal glands and NBRE; lane 6, as in lane 5, with the addition of antibodies directed against Nur77; lanes 7 and 8, as in lane 5, with the addition of a 10- or 25-fold excess of unlabeled NBRE, respectively. c, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 1, As in a and b. Lane 9, Protein extracts from untransfected COS cells in the presence of NurRE; lane 10, binding between extracts from pSG5-Nor1 transfected COS cells and NurRE; lane 11, binding between extracts from pSG5-Nur77 transfected COS cells and NurRE; lane 12, as in lane 11, with the addition of a 30-fold excess of unlabeled NurRE. The short arrows indicate the specific monomeric NBRE-Nor1 complexes; the long arrows indicate the specific monomeric NBRE-Nur77 or NurRE-Nur77 complexes. The open long arrow indicates the specific dimeric NurRE-Nur77 complex. The arrowhead indicates the mobility of the supershifted complex obtained in the presence of antibodies directed against Nur77. The two Nor1-DNA complexes detected in the gel shift experiments may be explained by the existence of differentially phosphorylated Nor1 populations. It is known that the DNA-binding activity of transcription factors can be modulated by phosphorylation (28). The treatment of COS protein extracts with calf intestinal phosphatase, abolished Nor1 binding, indicating that phosphorylation is necessary for its interaction with the DNA (data not shown). In a parallel experiment, gel retardation assays using COS protein extracts transfected with Nur77 and treated with the enzyme showed a Nur77-DNA-specific interaction. This agrees with the fact that Nur77 binds DNA and activates transcription when Ser354, present in its DNA-binding domain, is unphosphorylated (29, 30). Recently, it was reported that Nur77 interacts as a homodimer with a novel response element called NurRE, present in the POMC promoter. This site is composed of two octamers loosely related to NBRE and arranged in an inverse orientation separated by 6 bp (9). As shown in Fig. 4c, Nor1 did not interact as a homodimer with this palindromic DNA motif, even after the nuclear extract concentration was increased to a 3.5-fold excess. Two DNA-protein complexes similar to those observed for Nor1 and NBRE were detected only after overexposure of the gel to autoradiographic film (data not shown). Thus, Nor1, like Nur77, interacts with DNA as a monomer through its interaction with NBRE sequences. In contrast to Nur77, Nor1 failed to form homodimers capable of interacting with the palindromic NurRE, but did bind weakly to the NBRE-like sequences present in each half-site of the NurRE site. Nor1 activates the expression of a reporter gene under the control of the P450c21 promoter Analysis of the promoter elements of genes involved in steroidogenesis identified sequences containing the nuclear receptor half-site AGGTCA. In particular, the −72/−65 sequence of the P450c21 promoter contains a canonical NBRE site (31). Gel retardation assays showed that protein extracts from COS cells transfected with Nor1 interact with an oligonucleotide containing the P450c21 NBRE site (Fig. 5a). This indicated that Nor1 could also participate in the induction of P450c21 gene expression, consistent with the mRNA expression profile of both genes (see above). To further explore the role of Nor1 in the transcriptional regulation of the P450c21 promoter we analyzed first the ability of the protein to affect the enhancing capacity of the −72/−65 element. Y1 cells were transfected with the p−72/−65 P450c21-TK-CAT vector containing two copies of the −72/− 65 element (see Materials and Methods), and coexpression of Nor1 augmented significantly the activity of the reporter gene (Fig. 5b). Having established that Nor1 activates transcription from the −72/−65 sequence, we investigated its effect on the −150/+9 promoter region of the P450c21 gene. Cotransfection experiments with different concentrations of the Nor1 expression vector were performed in Y1 (data not shown) or C33 cells and showed that Nor1 increased the activity of a reporter gene under the control of the P450c21 promoter (Fig. 5c). Thus, Nor1 binds directly to the −72/−65 promoter element of the P450c21 gene and is able to activate this promoter in a dose-response manner. Figure 5. Open in new tabDownload slide Nor1 binds the −72/−65 P450c21 promoter element and activates the expression of a reporter gene under the control of the −150/+9 promoter region of the P450c21 gene. a, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 0, Protein extracts from untransfected COS cells in the presence of the −72/−65 P450c21 oligonucleotide; lane 1, binding between extracts from pSG5-Nor1-transfected COS cells and the −72/−65 P450c21 oligonucleotide; lanes 2 and 3, as in lane 1, with the addition of a 10- or 25-fold excess of unlabeled oligonucleotide, respectively; lane 4, as in lane 1, with the addition of a 25-fold excess of an unlabeled heterologous oligonucleotide MATE. b, Y1 cells were transfected with the −72/−65 P450c21-TK-CAT plasmid, and cotransfection with the pSG5-Nor1 expression vector increased the activity of the reporter gene. c, C33 cells were transfected with the p5′-P450c21-CAT plasmid, and cotransfection with the pSG5-Nor1 expression vector augmented the activity of the reporter gene in a dose-response manner. Figure 5. Open in new tabDownload slide Nor1 binds the −72/−65 P450c21 promoter element and activates the expression of a reporter gene under the control of the −150/+9 promoter region of the P450c21 gene. a, 5′-End-labeled double stranded oligonucleotides were incubated with protein extracts from COS cells that had been transfected with the pSG5-Nor1 plasmid. Lane 0, Protein extracts from untransfected COS cells in the presence of the −72/−65 P450c21 oligonucleotide; lane 1, binding between extracts from pSG5-Nor1-transfected COS cells and the −72/−65 P450c21 oligonucleotide; lanes 2 and 3, as in lane 1, with the addition of a 10- or 25-fold excess of unlabeled oligonucleotide, respectively; lane 4, as in lane 1, with the addition of a 25-fold excess of an unlabeled heterologous oligonucleotide MATE. b, Y1 cells were transfected with the −72/−65 P450c21-TK-CAT plasmid, and cotransfection with the pSG5-Nor1 expression vector increased the activity of the reporter gene. c, C33 cells were transfected with the p5′-P450c21-CAT plasmid, and cotransfection with the pSG5-Nor1 expression vector augmented the activity of the reporter gene in a dose-response manner. Second messenger pathways mediating Nor1 transcription in fasciculata adrenal cells Both forskolin and TPA induced Nor1 gene expression in primary cultures of fasciculata adrenal cells (Fig. 6a), and this induction was additive in the presence of both compounds. In all cases, the induction peak was attained within 2 h of treatment, indicating that expression of Nor1 in adrenal cells depends on protein kinase A (PKA) and protein kinase C (PKC) pathways. Figure 6. Open in new tabDownload slide Nor1 expression is induced by forskolin (F) or TPA (T) in adrenal fasciculata cells and by (FT) simultaneous treatment in Y1 cells. Northern blot analysis of RNAs obtained from bovine fasciculata cells (a) or Y1 cells (b) and (c). B, The experiment with T is not shown. In this experiment, as in the treatment with F, no induction of Nor1 expression was detected. Lane 0, Untreated cells; lanes 1–4, fasciculata cells treated for 45 min, 90 min, 150 min, and 4 h; lanes 5–7, Y1 cells treated for 30 min, 1 h, and 4 h. Figure 6. Open in new tabDownload slide Nor1 expression is induced by forskolin (F) or TPA (T) in adrenal fasciculata cells and by (FT) simultaneous treatment in Y1 cells. Northern blot analysis of RNAs obtained from bovine fasciculata cells (a) or Y1 cells (b) and (c). B, The experiment with T is not shown. In this experiment, as in the treatment with F, no induction of Nor1 expression was detected. Lane 0, Untreated cells; lanes 1–4, fasciculata cells treated for 45 min, 90 min, 150 min, and 4 h; lanes 5–7, Y1 cells treated for 30 min, 1 h, and 4 h. In contrast to Nur77 (Fig. 6c), Nor1 expression was not induced by ACTH, (Bu)2cAMP, forskolin, or TPA in the adrenocortical tumor cells Y1. Its expression was strongly induced only when these cells were treated with combinations of (Bu)2cAMP and TPA, or forskolin and TPA (Fig. 6b). Thus, the mechanisms of transcriptional activation of Nor1 and Nur77 appear to be different in Y1 cells. The differences observed between the induction of Nor1 transcription in primary cultures of normal fasciculata adrenal cells vs. the tumor cell line Y1, strongly suggest that in the latter case, factors essential for transcription are activated only when adenyl cyclase agonists and TPA act simultaneously. Discussion Nor1 is the most recent member of the NGFI-B/Nur77 subfamily to be cloned (1), and most of its properties are still unknown. In this study we analyze the trans-activation and DNA-binding properties of the nuclear protein, as well as some aspects of the regulation of the expression of its gene. ACTH stimulates the transcriptional activation of steroidogenic enzymes and regulates glucocorticoid synthesis. Its effects are indirect and are mediated by the cAMP pathway and proteins, which are most likely transcription factors (32). ACTH rapidly induces Nur77 mRNA synthesis in adrenal glands, and it has been shown that, in transfected cells, the receptor increases the activity of the P450c21 promoter through its interaction with the NBRE sequence. This DNA motif is also present in the promoters of genes encoding other steroidogenic enzymes (31, 33). These observations strongly implicate Nur77 as an intermediary in ACTH activity that ultimately results in increased steroidogenesis (11). In this report, we determine that ACTH can also induce the transcriptional activation of Nor1 in primary cultures of adrenal fasciculata cells. AngII is another hormone that controls adrenal steroidogenesis in vivo. It has been demonstrated that, in addition to regulating aldosterone production in glomerulosa cells, AngII has a direct stimulatory effect on cortisol synthesis in cultured fasciculata cells and a trophic effect on ACTH receptor number and ACTH responsiveness (24, 25). Here we show that AngII also induces the expression of Nor1 in cultured cells. We further demonstrate that Nor1, through its interaction with NBRE sequences, increases the activity of the P450c21 promoter in transfected cells. These data and the analysis of the time-course effects of ACTH and AngII on the expression of genes encoding steroidogenic enzymes, strongly suggest that Nor1 acts as an intermediate in the action of both hormones and is involved in their long-term effects. Sequence homology demonstrates that these nuclear receptors share a modular structure composed of several regions, named A/B, C, D, E, and F (26); the last one is absent in some receptors, including Nur77. The evolutionary conserved regions, C and E, contain the central zinc finger structure of the DNA-binding domain and the carboxyl putative ligand-binding domain, respectively. Two trans-activation functions have been attributed to region AF-1, located in the hypervariable N-terminal domain A/B, and to AF-2, located in region E (26). Our results indicate that an important activation domain is present in the amino-terminus of the Nor1 protein (amino acid residues 1–118). It has been shown that the sequence in the Nur77 A/B region that is important for its transcriptional activity, corresponds to N-54STFMDGYTGEFDTFLYQ70-C, a motif called TAB-1, enriched in hydrophobic and serine/threonine residues (34). The Nor1 sequence shows only 25% identity over its entire A/B region compared with the same region of Nur77 (1). Interestingly, the only sequence of Nor1 that is homologous to the Nur77 A/B region corresponds to TAB-1 (i.e. N-55STFMEGYPSSCELKPSCLYQ74-C). This finding strongly suggests that a TAB-I like sequence must also be important for Nor1 transcriptional activity. A region containing 14 histidines, 4 glutamines, and 1 proline, located between amino acid residues 99–118, link the AF-1 trans-activation domain of Nor1 to the rest of the molecule (1). Deletion analysis has also revealed that the profile of transcriptional activity mediated by the Nor1 carboxyl-terminal domain is also similar to that of Nur77 (34). Indeed, deletion of sequence 532–628 results in a significant decrease in transcriptional activity. However, deletion of sequence 409–532 partially restores the activity, suggesting that either the latter deletion is accompanied by a conformational change in the truncated protein or that the 409–532 sequence interacts with an inhibitory protein, and deletion of this region partially restores Nor1 activity. These results in addition to the 91% identity observed in their DNA-binding domains, strongly suggest that the overall structural and functional organization of Nor1 is similar to that of Nur77. In contrast to Nur77, it has been previously demonstrated that Nor1 is unable to form heterodimers with RXR (8), whereas a mechanism for Nur77 action was recently reported that involves formation of homodimers and interaction with a DNA palindromic motif called NurRE (9). Both halves of the NurRE site are required for the responsiveness to physiological signals in pituitary-derived cells. In the present report we show that Nor1 fails to bind as a homodimer to the palindromic motif present in the POMC promoter. In conclusion, the induction of Nor1 expression by ACTH and AngII, and its interaction with NBRE sequences present in the gene promoters of steroidogenic enzymes indicate that, like Nur77, Nor1 participates in the induction of the genes encoding these enzymes in adrenal cortex cells. In contrast, the inability of the protein to interact with the palindromic NurRE site by forming homodimers, may indicate that Nor1 cannot substitute for Nur77 function in the pituitary gland. During the submission of this manuscript, Maira et al. (35) indicated that the NurRE site of the POMC promoter is preferentially bound and activated by Nur77 homodimers. They also proposed that Nor1 may participate in the activation of the POMC gene essentially by a mechanism involving protein-protein interactions with Nur77. All of these data suggest that Nor1 has functions in the HPA axis that, compared with those of Nur77, are partly unique and partly redundant. In primary cultures of fasciculata cells, both forskolin and TPA can induce Nor1 expression. The effect of this induction is additive when the cells are treated with the two compounds together. ACTH also induces Nor1 expression in the same cells, and its action is known to be mediated by an increase in cAMP levels. AngII also stimulates Nor1 expression and may exert its effects through the activation of both branches of the phosphoinositide pathway, PKC and Ca2+/calmodulin (36–38). Recent experiments show that the stimulatory effect of AngII on early-immediate gene mRNA levels in fasciculata adrenal cells is mainly exerted through the action of PKC (39). In Y1 tumor cells, Nor1 expression was strongly induced when cells were treated with combinations of (Bu)2cAMP and TPA, or forskolin and TPA. Therefore, Nor1 expression in adrenal cortex cells also appears to depend on the PKA and PKC pathways. It is well established that activation of the cAMP-dependent PKA pathway by adenyl cyclase agonists results in phosphorylation and activation of the cAMP response element-binding protein and its subsequent binding to a promoter and the corresponding gene expression. It is also well known that the AP-1 complexes, which are induced by TPA, activate TPA-responsive elements. 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TI - Nuclear Receptors Nor1 and NGFI-B/Nur77 Play Similar, Albeit Distinct, Roles in the Hypothalamo-Pituitary-Adrenal Axis
JF - Endocrinology
DO - 10.1210/endo.141.7.7562
DA - 2000-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/nuclear-receptors-nor1-and-ngfi-b-nur77-play-similar-albeit-distinct-eqtBf00iLe
SP - 2392
VL - 141
IS - 7
DP - DeepDyve
ER -