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Molecular Cloning of Rat efp: Expression and Regulation in Primary Osteoblasts
Biochemical and Biophysical Research Communications 261, 412– 418 (1999) Article ID bbrc.1999.0874, available online at http://www.idealibrary.com on
Molecular Cloning of Rat efp: Expression and Regulation in Primary Osteoblasts Satoshi Inoue,* ,† Tomohiko Urano,† Sumito Ogawa,* ,† Tomoyuki Saito,† Akira Orimo,* Takayuki Hosoi,† Yasuyoshi Ouchi,† and Masami Muramatsu* ,1 *Department of Biochemistry, Saitama Medical School, 38 Moro-Hongo, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan, and †Department of Geriatric Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Received May 20, 1999
We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), by genomic binding-site cloning method. Here, we isolated a rat homologue of efp cDNA that encodes an open reading frame of 644 amino acids sharing high homology with human efp (69% identity at the protein level) and mouse efp (80% identity at the protein level). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box and B2 box, each having a pair of zinc fingers, and coiled-coil domain, belonging to the RING finger-B box-Coiled Coil (RBCC) family. Several members of RBCC family including efp have characteristic C-terminal domain, forming a subfamily. Next, we detected efp mRNA in primary osteoblasts, one of estrogen target cells, derived from the calvariae of rat fetus. An anti-efp antibody revealed the efp protein is expressed and regulated by estrogen in the primary osteoblasts. Interestingly, the efp protein in primary osteoblasts is down-regulated by 1a,25-dihydroxyvitamin D 3 treatment that promotes the differentiation of the cells, whereas it is up-regulated by TGF-b1 treatment that inhibits the differentiation of the cells. These findings suggest the possible involvement of the efp in the differentiation of osteoblastic cells. © 1999 Academic Press
Estrogen plays important roles not only in the female reproductive system but also in the nonreproductive system including the skeletal system. Osteoporosis is characterized by a reduction in bone mass and increased susceptibility to fractures. Estrogen deficiency is a cause of postmenopausal osteoporosis and estrogen replacement therapy is effective in prevention and treatment of bone loss (1–3). Estrogen actions are assumed to be mediated by estrogen receptor (ER), a member of nuclear receptor superfamily, that acts as 1
To whom reprint requests should be addressed. Fax: 81-492-949751. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
an estrogen-dependent transcription factor recognizing and binding to specific estrogen responsive element (ERE) in the enhancer region of target genes (4 – 6). ER regulates their transcription directly and exerts its action in the target cells. Recent studies demonstrate that both ERa and ERb (7) are present in human and rat osteoblastic cells (8, 9). To uncover molecular mechanisms of estrogen action, we have screened estrogen responsive genes by genomic binding-site cloning (10, 11). By the screening, we isolated the estrogen-responsive finger protein (efp) using a recombinant protein of DNA binding domain of ER (12). The efp is predominantly expressed in female reproductive organs including uterus, ovary and mammary gland (13). The efp gene has an ERE at the 39-untranslated region (UTR) and its expression is induced by estrogen in the uterus, brain and mammary gland cells (12, 13). The efp protein contains a RING finger, B box and coiled-coil domains characteristic of RING finger-B box-Coiled Coil (RBCC) family, of which members are supposed to be involved in transcriptional regulation, differentiation and oncogenesis (14). Therefore, we have inferred that the efp expression may be regulated during cell growth/differentiation and oncogenic processes of estrogen target cells. In this report, we describe the structure of rat efp and comparison among species as well as RBCC family members. Moreover, the expression and regulation of efp in rat primary osteoblasts during differentiation process have also been studied. MATERIALS AND METHODS Screening of a rat brain cDNA library and DNA sequence analysis. A lZAPII (Stratagene) cDNA library prepared from poly(A) 1 RNA of the rat brain was screened as described previously (12). 600,000 plaques were screened by hybridization with the 32P-labeled 0.5 kb Eco RI fragment of mouse efp cDNA (13). Then, the longest insert found in clone g7 was sequenced by dideoxy nucleotide chain termination method according to the manufacturer’s instruction (Sequenase, US Biochemical).
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Cell culture. Primary rat osteoblastic cells were isolated from calvariae of 21 day old Sprague-Dawley rat embryos as described previously (15). The cells were maintained in a-MEM containing 10% FCS and antibiotics. Cells at the second passage were used for all experiments. Reverse-transcription PCR (RT-PCR). cDNA was synthesized from 0.1 mg of rat poly(A) 1RNA of primary osteoblasts using random 9 mers and AMV reverse transcriptase (TaKaRa). Subsequent PCR amplification was carried out by the RNA PCR kit (TaKaRa) for 30 cycles using an annealing temperature of 55°C in a Perkin Elmer thermalcycler (Perkin Elmer Cetus). The oligonucleotides, 59-GCAAAGCACTGGAGGATG-39 and 59-TTGTCCAGGAGCTCCAAAGG-39 were used for amplification of 643 bp fragment of rat efp mRNA. The oligonucleotide sets, 59-CTCTTGGACAGGAATCAAGG39/59-TAGAGAGGCACGACATTCTT-39 and 59-CATCAGTAACAAGGGCATGG-39/59-CACTGAGACTGTAGGTTCTG-39 were used for amplification of 386 bp fragment of rat ERa mRNA (16) and 192 bp fragment of ERb mRNA (7), respectively. The plasmids containing rat efp, ERa (16) and ERb (7) cDNA were used for positive controls of PCR reactions. Alkaline phosphatase (ALP) activity. Cells were plated and maintained in 6-well plastic dishes as described below. After serum starvation, cells were treated with 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml) for 48 h. The cells were sonicated 0.1 M Tris buffer, pH 7.2, containing 0.1% Triton X-100. ALP activity was determined using p-nitro-phenylphosphate as a substrate in 0.05 M 2-amino-2-methylpropanol and 2 mM MgCl 2, pH 10.5. The amount of p-nitro-phenol released was determined by measuring absorbance at 410 nm. ALP activity was corrected for protein content which was determined with the Bio-Rad protein assay reagent (Bio-Rad). Antibodies and immunological methods. Affinity-purified antiefp antibody was prepared (13). In vitro translation was performed with pBluescript-g7 or pBluescript (for mock) using TNT rabbit reticulocyte lysate according to manufacturer’s instruction (Promega). The antibody cross-reacts efficiently with the rat homologue. Cells were plated in 6-well plastic dishes with a-MEM containing 10% FCS, and were cultured in a-MEM containing 0.5% FCS for 72 h prior to the experiment. Then the cells were treated with 17b-estradiol (10 nM) for 2, 4, 8 or 12 h and with 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml) for 24 or 48 h. Cells were rinsed twice with ice-cold PBS and lysed in 180 ml Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 5 mM EGTA, 2 mM sodium vanadate, 0.5% sodium deoxycholate 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 0.1% Nonidet P-40], and the lysates were cleared by centrifugation at 15,000 g for 5 min at 4°C. For immunoblot analysis, the samples were separated on 7.5% SDS-PAGE. Western blotting was performed using enhanced chemiluminescence detection system (Amersham).
RESULTS Isolation of Rat efp cDNA To isolate rat efp cDNA, a lZAP II cDNA library prepared from poly(A) 1 RNA of rat brain was screened with 0.5 kb EcoRI fragment of mouse efp cDNA. Sixteen clones out of 600,000 plaques were found positive and all the clones were indicated that they were derived from the same RNA by restriction mapping and partial sequencing. The clone g7 that had the longest insert was completely sequenced and it had the longest open reading frame of 644 amino acids, showing a high degree of homology to the predicted mouse and human efp protein. The calculated relative molecular mass (Mr) of the predicted rat efp protein is 721,152.
FIG. 1. Nucleotide and deduced amino acid sequences of rat efp cDNA. The deduced amino acids are shown below their respective codons. The TAG stop codon is indicated by asterisk. Conserved residues, including cysteines/histidines, in RING finger and B1/B2 box domains, which are involved in zinc finger-like structures are circled. The potential coiled-coil and C-terminal domains are underlined.
Structure of Rat efp Protein Rat efp cDNA encodes a protein containing the RING finger motif, B1 box, B2 box, coiled-coil and C-terminal domain (Fig. 1). Alignment of the predicted human, rat and mouse efp proteins (Fig. 2A) shows that they are
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FIG. 2. Sequence alignment and comparison among human, rat and mouse efp proteins. (A) Sequence alignment of human, rat and mouse efp. The conserved amino acids with rat are shown by dots. The gap sequence is indicated by “–”. (B) Diagrammatic representation and comparison of conserved domains among human, rat, and mouse efp. The RING finger, B1 box, B2 box, coiled-coil, and C-terminal domains are shown as distinctive boxes. The homology (%) of amino acids between rat and human efp (upper) and between rat and mouse efp (lower) are shown for respective conserved domains.
very similar in RING finger (90% with human and 90% with mouse), B1 box (92% with human and 92% with mouse), B2 box (90% with human and 90% with mouse), and C-terminal domain (85% with human and 92% with mouse) at the amino acids
level (Fig. 2B). The spacing between coiled-coil domain and C-terminal domain shows low homology to that of human and mouse efp. In Fig. 3, the rat efp is also compared with other members of the RBCC family (14).
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FIG. 3. Schematic representation of the members of RBCC family. Diagram of domain structure of RBCC family. Several members of RBCC family have the C-terminal domain conserved among them, forming a subfamily. RING finger, B-boxes, coiled-coil, and C-terminal domains are shown by closed oval, hatched oval, open rectangle, and hatched rectangle, respectively.
The Expression and Regulation of efp in Rat Primary Osteoblasts RT-PCR analysis showed the presence of ERa, ERb and efp mRNA in rat primary osteoblasts (Fig. 4A). Then, Western blot analysis also detected the presence of natural efp protein in these cells (Fig. 4B). The size of the natural product (70 kD) agreed with the predicted M r and the size of the band detected from in vitro translation products. The intensity of signal detected by the anti-efp antibody increased after estrogen treatment within 2 h and reached a peak at 8 h (Fig. 4C). 1a,25-Dihydroxyvitamin D 3 and TGF-b modulate growth and differentiation states of primary osteo-
blasts. ALP activities after 1a,25-dihydroxyvitamin D 3 and TGF-b1 treatment for 48 h were compared in Fig. 5A. ALP activity increased by 1a,25-dihydroxyvitamin D 3, whereas it decreased by TGF-b1. Western blotting indicated that the intensity of the band detected by anti-efp antibody decreased by 1a,25-dihydroxyvitamin D 3 and increased by TGF-b1 within 24 h. DISCUSSION In the present study, the rat homologue of efp has been isolated and characterized. Human efp has been identified as an estrogen-responsive gene by isolating
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FIG. 4. epf as an estrogen responsive gene expressed in rat primary osteoblasts. (A) RT-PCR analysis from poly(A) 1 RNA of rat primary osteoblasts using specific primers for efp, ERa and ERb. Expression of efp, ERa and ERb transcripts (643 bp, 386 bp, 192 bp, respectively) was analyzed by RT-PCR with 0.1 mg of poly(A) 1 RNA. The RT-PCR products were analyzed by agarose gel electrophoresis. (B) Western blot analysis of the efp protein in the rat primary osteoblasts. Cell extracts prepared from the rat primary osteoblasts (lane 3) and in vitro translation products with (lane 2) and without (lane 1) rat efp plasmid were analyzed by Western blotting. The 70 kD band, the size of which agreed with the natural products in primary osteoblasts, was detected in in vitro translation products with the rat efp plasmid. (C) The efp protein is regulated by estrogen. Cell extracts (20 mg) were prepared from the rat primary osteoblasts at indicated h after 17b-estradiol (10 nM) treatment and analyzed by immunoblotting using the anti-efp antibody.
binding fragments from human genomic DNA using a recombinant ER protein (12). To understand the mechanism of action of a transcription factor, it is indispensable to know its target genes in vivo. Because steroid/ thyroid hormone receptors and retinoic acids receptors are transcription factors, this approach can be applicable to identify their targets and to clarify mechanisms of their actions. Here, we have demonstrated that the efp is an estrogen responsive gene also in the rat, confirming the usefulness of genomic binding-site cloning. The predicted rat efp protein shows a high degree of conservation with mouse and human efp over the RING finger, B boxes, coiled-coil and C-terminal domains, diverging significantly only at their spacing between coiled-coil and C-terminal domain. A number of proteins having RBCC domains are supposed to be involved in regulating gene expression, cell differentiation, oncogenesis and several diseases (14). For example, XNF-7 is a putative transcription regulator expressed maternally in Xenopus laevis (17). PWA33 is associated with the nascent transcripts on the lampbrush chromosome loops and may function as a regulatory protein during early development (18). The component of human SS-A/Ro is an autoantigen in Sjo¨gren’s syndrome and systemic lupus erythematosus (19, 20). MID1 is found disrupted or mutated in patients of Opitz G/BBB syndrome, an inherited disorder
characterized by midline defects including hypertelorism, hypospadias, lip-palate-laryngotracheal clefts and imperforate anus (21). Human rfp is fused with the ret proto-oncogene in a transforming protein resulting from a chromosomal translocation (22). Both rfp and terf (23) mRNA are predominantly expressed in the testis. These proteins mentioned above together with the efp have the C-termainal domain, forming a subfamily of RBCC family. The C-terminal domain among human, rat and mouse efp is highly conserved and it is assumed that this domain perform significant functions. It is notable that mutaions of MID1 clustered in the C-terminal domain of the MID1 protein, suggesting that this conserved domain of RBCC proteins play a critical role in the pathogenesis of Opitz syndrome and in midline developments during blastogenesis (21, 24). Some RBCC proteins such as TIF1 and PML do not possess the C-terminal domain. TIF1 originally found as a transforming mouse fusion protein with the B-Raf proto-oncogene (25), was reported to be a mediator of the ligand-dependent activation of nuclear receptors (26). PML is the counterpart of a fusion protein with the retinoic acid receptor a (RARa) in acute promyelocytic leukemia (APL) (27–29). We have previously shown that the efp mRNA is detected in ovary, uterus, mammary gland and placenta at higher levels by RNase protection assay (13). In situ hybridization histochemistry has also revealed
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FIG. 5. Expression of efp protein in rat primary osteoblasts treated with 1a,25-dihydroxyvitamin D 3 and TGF-b1. (A) 1a,25Dihydroxyvitamin D 3 enhances and TGF-b1 inhibits differentiation of primary osteoblasts. ALP activities were measured as a marker of differentiation and shown by relative activity (% control) after 48 h treatment of 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml). Values are expressed as mean 6 SEM. *p , 0.05 in comparison with control. (B) Western blot analysis was performed with cell extracts from primary osteoblasts. Cell extracts (20 mg) were prepared before treatment (lane 1) and after treatment without ligand (lane 2, 24 h; lane 3, 48 h), with 1a,25-dihydroxyvitamin D 3 (10 nM) (lane 4, 24 h; lane 5, 48 h) or with TGF-b1 (10 ng/ml) (lane 6, 24 h; lane 7, 48 h). The intensity of the signal detected by the anti-efp antibody decreased after 1a,25-dihydroxyvitamin D 3 treatment and increased after TGF-b1 treatment.
that the efp mRNA and ERa mRNA are co-localized in uterus, ovary, mammary gland and certain regions in the brain (13). Here, we studied expression of efp in rat primary osteoblasts that are derived from the bone, one of the estrogen target organs. RT-PCR confirmed the existence of ERa and ERb mRNA in these cells. The expression of efp at the mRNA level and the protein level was clearly shown by RT-PCR and Western blotting, respectively. We also confirmed the expression of efp in rat osteoblastic cells derived from osteosarcoma, ROS17/2.8, by the same method (data not shown). Although it is well-known that osteoblasts are targets for estrogen, estrogen responsive genes identified in these cells are few. In the present study, we have demonstrated that the efp is one of the estrogen responsive genes in osteoblasts. Primary rat osteoblasts are being used for a good model of cell growth and differentiation (30). 1a,25Dihydroxyvitamin D 3 inhibits growth and promotes differentiation of primary osteoblasts (31). TGF-b promotes growth and inhibits differentiation of these cells (32). In line with previous reports, ALP activity, a marker of osteoblastic differentiation, increased by 1a,25-dihydroxyvitamin D 3, but decreased by TGF-b1. During these treatments, the expression of efp was down-regulated by 1a,25-dihydroxyvitamin D 3 and upregulated by TGF-b1. The genes that are regulated in the course of differentiation of osteoblasts should be important to understand the function of the skeletal system. They will also contribute to the development of
drugs for osteoporosis. Further studies are required to reveal the roles of the efp in osteoblast growth and differentiation. ACKNOWLEGMENTS We thank Dr. J. Å. Gustafsson for the gift of rat ERb plasmid and Ms. M. Kobayashi for expert technical assistance. This work was supported in part by research grants from the comprehensive Research on Aging and Health, the Ministry of Health and Welfare of Japan, KANZAWA Medical Research Foundation and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST).
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Molecular Cloning of Rat efp: Expression and Regulation in Primary Osteoblasts Satoshi Inoue,* ,† Tomohiko Urano,† Sumito Ogawa,* ,† Tomoyuki Saito,† Akira Orimo,* Takayuki Hosoi,† Yasuyoshi Ouchi,† and Masami Muramatsu* ,1 *Department of Biochemistry, Saitama Medical School, 38 Moro-Hongo, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan, and †Department of Geriatric Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
Received May 20, 1999
We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), by genomic binding-site cloning method. Here, we isolated a rat homologue of efp cDNA that encodes an open reading frame of 644 amino acids sharing high homology with human efp (69% identity at the protein level) and mouse efp (80% identity at the protein level). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box and B2 box, each having a pair of zinc fingers, and coiled-coil domain, belonging to the RING finger-B box-Coiled Coil (RBCC) family. Several members of RBCC family including efp have characteristic C-terminal domain, forming a subfamily. Next, we detected efp mRNA in primary osteoblasts, one of estrogen target cells, derived from the calvariae of rat fetus. An anti-efp antibody revealed the efp protein is expressed and regulated by estrogen in the primary osteoblasts. Interestingly, the efp protein in primary osteoblasts is down-regulated by 1a,25-dihydroxyvitamin D 3 treatment that promotes the differentiation of the cells, whereas it is up-regulated by TGF-b1 treatment that inhibits the differentiation of the cells. These findings suggest the possible involvement of the efp in the differentiation of osteoblastic cells. © 1999 Academic Press
Estrogen plays important roles not only in the female reproductive system but also in the nonreproductive system including the skeletal system. Osteoporosis is characterized by a reduction in bone mass and increased susceptibility to fractures. Estrogen deficiency is a cause of postmenopausal osteoporosis and estrogen replacement therapy is effective in prevention and treatment of bone loss (1–3). Estrogen actions are assumed to be mediated by estrogen receptor (ER), a member of nuclear receptor superfamily, that acts as 1
To whom reprint requests should be addressed. Fax: 81-492-949751. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
an estrogen-dependent transcription factor recognizing and binding to specific estrogen responsive element (ERE) in the enhancer region of target genes (4 – 6). ER regulates their transcription directly and exerts its action in the target cells. Recent studies demonstrate that both ERa and ERb (7) are present in human and rat osteoblastic cells (8, 9). To uncover molecular mechanisms of estrogen action, we have screened estrogen responsive genes by genomic binding-site cloning (10, 11). By the screening, we isolated the estrogen-responsive finger protein (efp) using a recombinant protein of DNA binding domain of ER (12). The efp is predominantly expressed in female reproductive organs including uterus, ovary and mammary gland (13). The efp gene has an ERE at the 39-untranslated region (UTR) and its expression is induced by estrogen in the uterus, brain and mammary gland cells (12, 13). The efp protein contains a RING finger, B box and coiled-coil domains characteristic of RING finger-B box-Coiled Coil (RBCC) family, of which members are supposed to be involved in transcriptional regulation, differentiation and oncogenesis (14). Therefore, we have inferred that the efp expression may be regulated during cell growth/differentiation and oncogenic processes of estrogen target cells. In this report, we describe the structure of rat efp and comparison among species as well as RBCC family members. Moreover, the expression and regulation of efp in rat primary osteoblasts during differentiation process have also been studied. MATERIALS AND METHODS Screening of a rat brain cDNA library and DNA sequence analysis. A lZAPII (Stratagene) cDNA library prepared from poly(A) 1 RNA of the rat brain was screened as described previously (12). 600,000 plaques were screened by hybridization with the 32P-labeled 0.5 kb Eco RI fragment of mouse efp cDNA (13). Then, the longest insert found in clone g7 was sequenced by dideoxy nucleotide chain termination method according to the manufacturer’s instruction (Sequenase, US Biochemical).
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Cell culture. Primary rat osteoblastic cells were isolated from calvariae of 21 day old Sprague-Dawley rat embryos as described previously (15). The cells were maintained in a-MEM containing 10% FCS and antibiotics. Cells at the second passage were used for all experiments. Reverse-transcription PCR (RT-PCR). cDNA was synthesized from 0.1 mg of rat poly(A) 1RNA of primary osteoblasts using random 9 mers and AMV reverse transcriptase (TaKaRa). Subsequent PCR amplification was carried out by the RNA PCR kit (TaKaRa) for 30 cycles using an annealing temperature of 55°C in a Perkin Elmer thermalcycler (Perkin Elmer Cetus). The oligonucleotides, 59-GCAAAGCACTGGAGGATG-39 and 59-TTGTCCAGGAGCTCCAAAGG-39 were used for amplification of 643 bp fragment of rat efp mRNA. The oligonucleotide sets, 59-CTCTTGGACAGGAATCAAGG39/59-TAGAGAGGCACGACATTCTT-39 and 59-CATCAGTAACAAGGGCATGG-39/59-CACTGAGACTGTAGGTTCTG-39 were used for amplification of 386 bp fragment of rat ERa mRNA (16) and 192 bp fragment of ERb mRNA (7), respectively. The plasmids containing rat efp, ERa (16) and ERb (7) cDNA were used for positive controls of PCR reactions. Alkaline phosphatase (ALP) activity. Cells were plated and maintained in 6-well plastic dishes as described below. After serum starvation, cells were treated with 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml) for 48 h. The cells were sonicated 0.1 M Tris buffer, pH 7.2, containing 0.1% Triton X-100. ALP activity was determined using p-nitro-phenylphosphate as a substrate in 0.05 M 2-amino-2-methylpropanol and 2 mM MgCl 2, pH 10.5. The amount of p-nitro-phenol released was determined by measuring absorbance at 410 nm. ALP activity was corrected for protein content which was determined with the Bio-Rad protein assay reagent (Bio-Rad). Antibodies and immunological methods. Affinity-purified antiefp antibody was prepared (13). In vitro translation was performed with pBluescript-g7 or pBluescript (for mock) using TNT rabbit reticulocyte lysate according to manufacturer’s instruction (Promega). The antibody cross-reacts efficiently with the rat homologue. Cells were plated in 6-well plastic dishes with a-MEM containing 10% FCS, and were cultured in a-MEM containing 0.5% FCS for 72 h prior to the experiment. Then the cells were treated with 17b-estradiol (10 nM) for 2, 4, 8 or 12 h and with 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml) for 24 or 48 h. Cells were rinsed twice with ice-cold PBS and lysed in 180 ml Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 5 mM EGTA, 2 mM sodium vanadate, 0.5% sodium deoxycholate 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 0.1% Nonidet P-40], and the lysates were cleared by centrifugation at 15,000 g for 5 min at 4°C. For immunoblot analysis, the samples were separated on 7.5% SDS-PAGE. Western blotting was performed using enhanced chemiluminescence detection system (Amersham).
RESULTS Isolation of Rat efp cDNA To isolate rat efp cDNA, a lZAP II cDNA library prepared from poly(A) 1 RNA of rat brain was screened with 0.5 kb EcoRI fragment of mouse efp cDNA. Sixteen clones out of 600,000 plaques were found positive and all the clones were indicated that they were derived from the same RNA by restriction mapping and partial sequencing. The clone g7 that had the longest insert was completely sequenced and it had the longest open reading frame of 644 amino acids, showing a high degree of homology to the predicted mouse and human efp protein. The calculated relative molecular mass (Mr) of the predicted rat efp protein is 721,152.
FIG. 1. Nucleotide and deduced amino acid sequences of rat efp cDNA. The deduced amino acids are shown below their respective codons. The TAG stop codon is indicated by asterisk. Conserved residues, including cysteines/histidines, in RING finger and B1/B2 box domains, which are involved in zinc finger-like structures are circled. The potential coiled-coil and C-terminal domains are underlined.
Structure of Rat efp Protein Rat efp cDNA encodes a protein containing the RING finger motif, B1 box, B2 box, coiled-coil and C-terminal domain (Fig. 1). Alignment of the predicted human, rat and mouse efp proteins (Fig. 2A) shows that they are
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FIG. 2. Sequence alignment and comparison among human, rat and mouse efp proteins. (A) Sequence alignment of human, rat and mouse efp. The conserved amino acids with rat are shown by dots. The gap sequence is indicated by “–”. (B) Diagrammatic representation and comparison of conserved domains among human, rat, and mouse efp. The RING finger, B1 box, B2 box, coiled-coil, and C-terminal domains are shown as distinctive boxes. The homology (%) of amino acids between rat and human efp (upper) and between rat and mouse efp (lower) are shown for respective conserved domains.
very similar in RING finger (90% with human and 90% with mouse), B1 box (92% with human and 92% with mouse), B2 box (90% with human and 90% with mouse), and C-terminal domain (85% with human and 92% with mouse) at the amino acids
level (Fig. 2B). The spacing between coiled-coil domain and C-terminal domain shows low homology to that of human and mouse efp. In Fig. 3, the rat efp is also compared with other members of the RBCC family (14).
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FIG. 3. Schematic representation of the members of RBCC family. Diagram of domain structure of RBCC family. Several members of RBCC family have the C-terminal domain conserved among them, forming a subfamily. RING finger, B-boxes, coiled-coil, and C-terminal domains are shown by closed oval, hatched oval, open rectangle, and hatched rectangle, respectively.
The Expression and Regulation of efp in Rat Primary Osteoblasts RT-PCR analysis showed the presence of ERa, ERb and efp mRNA in rat primary osteoblasts (Fig. 4A). Then, Western blot analysis also detected the presence of natural efp protein in these cells (Fig. 4B). The size of the natural product (70 kD) agreed with the predicted M r and the size of the band detected from in vitro translation products. The intensity of signal detected by the anti-efp antibody increased after estrogen treatment within 2 h and reached a peak at 8 h (Fig. 4C). 1a,25-Dihydroxyvitamin D 3 and TGF-b modulate growth and differentiation states of primary osteo-
blasts. ALP activities after 1a,25-dihydroxyvitamin D 3 and TGF-b1 treatment for 48 h were compared in Fig. 5A. ALP activity increased by 1a,25-dihydroxyvitamin D 3, whereas it decreased by TGF-b1. Western blotting indicated that the intensity of the band detected by anti-efp antibody decreased by 1a,25-dihydroxyvitamin D 3 and increased by TGF-b1 within 24 h. DISCUSSION In the present study, the rat homologue of efp has been isolated and characterized. Human efp has been identified as an estrogen-responsive gene by isolating
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FIG. 4. epf as an estrogen responsive gene expressed in rat primary osteoblasts. (A) RT-PCR analysis from poly(A) 1 RNA of rat primary osteoblasts using specific primers for efp, ERa and ERb. Expression of efp, ERa and ERb transcripts (643 bp, 386 bp, 192 bp, respectively) was analyzed by RT-PCR with 0.1 mg of poly(A) 1 RNA. The RT-PCR products were analyzed by agarose gel electrophoresis. (B) Western blot analysis of the efp protein in the rat primary osteoblasts. Cell extracts prepared from the rat primary osteoblasts (lane 3) and in vitro translation products with (lane 2) and without (lane 1) rat efp plasmid were analyzed by Western blotting. The 70 kD band, the size of which agreed with the natural products in primary osteoblasts, was detected in in vitro translation products with the rat efp plasmid. (C) The efp protein is regulated by estrogen. Cell extracts (20 mg) were prepared from the rat primary osteoblasts at indicated h after 17b-estradiol (10 nM) treatment and analyzed by immunoblotting using the anti-efp antibody.
binding fragments from human genomic DNA using a recombinant ER protein (12). To understand the mechanism of action of a transcription factor, it is indispensable to know its target genes in vivo. Because steroid/ thyroid hormone receptors and retinoic acids receptors are transcription factors, this approach can be applicable to identify their targets and to clarify mechanisms of their actions. Here, we have demonstrated that the efp is an estrogen responsive gene also in the rat, confirming the usefulness of genomic binding-site cloning. The predicted rat efp protein shows a high degree of conservation with mouse and human efp over the RING finger, B boxes, coiled-coil and C-terminal domains, diverging significantly only at their spacing between coiled-coil and C-terminal domain. A number of proteins having RBCC domains are supposed to be involved in regulating gene expression, cell differentiation, oncogenesis and several diseases (14). For example, XNF-7 is a putative transcription regulator expressed maternally in Xenopus laevis (17). PWA33 is associated with the nascent transcripts on the lampbrush chromosome loops and may function as a regulatory protein during early development (18). The component of human SS-A/Ro is an autoantigen in Sjo¨gren’s syndrome and systemic lupus erythematosus (19, 20). MID1 is found disrupted or mutated in patients of Opitz G/BBB syndrome, an inherited disorder
characterized by midline defects including hypertelorism, hypospadias, lip-palate-laryngotracheal clefts and imperforate anus (21). Human rfp is fused with the ret proto-oncogene in a transforming protein resulting from a chromosomal translocation (22). Both rfp and terf (23) mRNA are predominantly expressed in the testis. These proteins mentioned above together with the efp have the C-termainal domain, forming a subfamily of RBCC family. The C-terminal domain among human, rat and mouse efp is highly conserved and it is assumed that this domain perform significant functions. It is notable that mutaions of MID1 clustered in the C-terminal domain of the MID1 protein, suggesting that this conserved domain of RBCC proteins play a critical role in the pathogenesis of Opitz syndrome and in midline developments during blastogenesis (21, 24). Some RBCC proteins such as TIF1 and PML do not possess the C-terminal domain. TIF1 originally found as a transforming mouse fusion protein with the B-Raf proto-oncogene (25), was reported to be a mediator of the ligand-dependent activation of nuclear receptors (26). PML is the counterpart of a fusion protein with the retinoic acid receptor a (RARa) in acute promyelocytic leukemia (APL) (27–29). We have previously shown that the efp mRNA is detected in ovary, uterus, mammary gland and placenta at higher levels by RNase protection assay (13). In situ hybridization histochemistry has also revealed
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FIG. 5. Expression of efp protein in rat primary osteoblasts treated with 1a,25-dihydroxyvitamin D 3 and TGF-b1. (A) 1a,25Dihydroxyvitamin D 3 enhances and TGF-b1 inhibits differentiation of primary osteoblasts. ALP activities were measured as a marker of differentiation and shown by relative activity (% control) after 48 h treatment of 1a,25-dihydroxyvitamin D 3 (10 nM) and TGF-b1 (10 ng/ml). Values are expressed as mean 6 SEM. *p , 0.05 in comparison with control. (B) Western blot analysis was performed with cell extracts from primary osteoblasts. Cell extracts (20 mg) were prepared before treatment (lane 1) and after treatment without ligand (lane 2, 24 h; lane 3, 48 h), with 1a,25-dihydroxyvitamin D 3 (10 nM) (lane 4, 24 h; lane 5, 48 h) or with TGF-b1 (10 ng/ml) (lane 6, 24 h; lane 7, 48 h). The intensity of the signal detected by the anti-efp antibody decreased after 1a,25-dihydroxyvitamin D 3 treatment and increased after TGF-b1 treatment.
that the efp mRNA and ERa mRNA are co-localized in uterus, ovary, mammary gland and certain regions in the brain (13). Here, we studied expression of efp in rat primary osteoblasts that are derived from the bone, one of the estrogen target organs. RT-PCR confirmed the existence of ERa and ERb mRNA in these cells. The expression of efp at the mRNA level and the protein level was clearly shown by RT-PCR and Western blotting, respectively. We also confirmed the expression of efp in rat osteoblastic cells derived from osteosarcoma, ROS17/2.8, by the same method (data not shown). Although it is well-known that osteoblasts are targets for estrogen, estrogen responsive genes identified in these cells are few. In the present study, we have demonstrated that the efp is one of the estrogen responsive genes in osteoblasts. Primary rat osteoblasts are being used for a good model of cell growth and differentiation (30). 1a,25Dihydroxyvitamin D 3 inhibits growth and promotes differentiation of primary osteoblasts (31). TGF-b promotes growth and inhibits differentiation of these cells (32). In line with previous reports, ALP activity, a marker of osteoblastic differentiation, increased by 1a,25-dihydroxyvitamin D 3, but decreased by TGF-b1. During these treatments, the expression of efp was down-regulated by 1a,25-dihydroxyvitamin D 3 and upregulated by TGF-b1. The genes that are regulated in the course of differentiation of osteoblasts should be important to understand the function of the skeletal system. They will also contribute to the development of
drugs for osteoporosis. Further studies are required to reveal the roles of the efp in osteoblast growth and differentiation. ACKNOWLEGMENTS We thank Dr. J. Å. Gustafsson for the gift of rat ERb plasmid and Ms. M. Kobayashi for expert technical assistance. This work was supported in part by research grants from the comprehensive Research on Aging and Health, the Ministry of Health and Welfare of Japan, KANZAWA Medical Research Foundation and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST).
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