Sensing of Extracellular Cations in CasR-deficient Osteoblasts
2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês
10.1074/jbc.275.5.3256
ISSN1083-351X
AutoresMin Pi, Sanford C. Garner, Patrick J. Flannery, Robert F. Spurney, L. Darryl Quarles,
Tópico(s)Parathyroid Disorders and Treatments
ResumoWe isolated osteoblastic cell lines from wild-type (CasR +/+) and receptor null (CasR −/−) mice to investigate whetherCasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from bothCasR +/+ and CasR −/−mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR −/− osteoblasts. The functional responses to these cations were identical inCasR +/+ and CasR −/−osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition,CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR +/+ orCasR −/− osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative forCasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts fromCasR −/− mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism. We isolated osteoblastic cell lines from wild-type (CasR +/+) and receptor null (CasR −/−) mice to investigate whetherCasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from bothCasR +/+ and CasR −/−mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR −/− osteoblasts. The functional responses to these cations were identical inCasR +/+ and CasR −/−osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition,CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR +/+ orCasR −/− osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative forCasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts fromCasR −/− mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism. reverse transcription-polymerase chain reaction base pair(s) serum response element Tris-buffered saline prostaglandin E1 CasR is a G-protein-coupled extracellular calcium-sensing receptor that was originally isolated and cloned from parathyroid (1.Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2384) Google Scholar), kidney (2.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar), and brain (3.Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (344) Google Scholar) cDNA libraries. The major physiological function of CasR is to transduce extracellular calcium regulation of parathyroid hormone secretion in parathyroid glands. This role is supported by the findings that inactivating mutations of CasR cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (NSHPT) (4.Pollak M.R. Brown E.M. Chou Y.Y.W. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (915) Google Scholar) and that activating mutations result in hypoparathyroidism (5.Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). In addition, targeted ablation of CasR results in hypercalcemia and elevated parathyroid hormone levels in mice (6.Ho C. Conner D.A. Pollak M.R. Ladd D.J. Kifor O. Warren H.B. Brown E.M. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 389-394Crossref PubMed Scopus (523) Google Scholar). The function ofCasR in other tissues is less clear. In the kidney,CasR may modulate renal tubular transport, possibly acting as an antagonist to parathyroid hormone actions on renal calcium handling (7.Brown E.M. Pollak M. Seidman J.G. Chou Y.H. Riccardi D. Hebert S.C. N. Engl. J. Med. 1995; 333: 234-240Crossref PubMed Scopus (0) Google Scholar). CasR is also located in nerve terminals, where it may transduce calcium-mediated neurotransmitter release (3.Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (344) Google Scholar).CasR is detected in low abundance in many other tissues and cell types, where its physiologic role is uncertain (7.Brown E.M. Pollak M. Seidman J.G. Chou Y.H. Riccardi D. Hebert S.C. N. Engl. J. Med. 1995; 333: 234-240Crossref PubMed Scopus (0) Google Scholar, 8.Pearce S.H.S. Trump D. Wooding C. Basser G.M. Chew S.L. Grant D.B. Heath D.A. Hughes I.A. Paterson C.R. Whythe M.P. Thakker R.V. J. Clin. Invest. 1995; 96: 2683-2692Crossref PubMed Scopus (330) Google Scholar, 9.Chattopadhyay N. Vassilev P.M. Brown E.M. Biol. Chem. 1997; 378: 759-768PubMed Google Scholar). Bone is one such tissue in which the expression of CasR and its physiologic role remain uncertain (10.House M.G. Kohlmeier L. Chattopadhyay N. Kifor O. Yamaguchi T. Leboff M.S. Glowacki J. Brown E.M. J. Bone Miner. Res. 1997; 12: 1959-1970Crossref PubMed Scopus (167) Google Scholar, 11.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar, 12.Kameda T. Mano H. Yamada Y. Takai H. Amizuka N. Kobori M. Izuma N. Kawashima H. Ozawa H. Ikeda K. Kameda A. Hakeda Y. Kumegawa M. Biochem. Biophys. Res. Commun. 1998; 245: 419-422Crossref PubMed Scopus (191) Google Scholar, 13.Yamaguchi T. Kifor O. Chattopadhyay N. Brown E.M. Biochem. Biophys. Res. Commun. 1998; 243: 753-757Crossref PubMed Scopus (87) Google Scholar, 14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar). AlthoughCasR is reported to be expressed in bone marrow cells (10.House M.G. Kohlmeier L. Chattopadhyay N. Kifor O. Yamaguchi T. Leboff M.S. Glowacki J. Brown E.M. J. Bone Miner. Res. 1997; 12: 1959-1970Crossref PubMed Scopus (167) Google Scholar,11.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar), there are conflicting data regarding its expression in cells within the osteoblast lineage (13.Yamaguchi T. Kifor O. Chattopadhyay N. Brown E.M. Biochem. Biophys. Res. Commun. 1998; 243: 753-757Crossref PubMed Scopus (87) Google Scholar, 14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar, 15.Demarest K.T. Minor L.K. Gunnet J.W. J. Bone Miner. Res. 1997; 12 Suppl. 1: S413Google Scholar, 16.Pi M. Hinson T.K. Quarles L.D. J. Bone Miner. Res. 1999; 14: 1310-1319Crossref PubMed Scopus (63) Google Scholar, 17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 18.Bapty B.W. Dai L.J. Ritchie G. Jirik F. Canaff L. Hendy G.N. Quamme G.A. Kidney Int. 1998; 53: 583-592Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 19.Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar). Some studies have reported the detection of CasR in osteoblasts by Western blot and RT-PCR1 analyses (13.Yamaguchi T. Kifor O. Chattopadhyay N. Brown E.M. Biochem. Biophys. Res. Commun. 1998; 243: 753-757Crossref PubMed Scopus (87) Google Scholar, 14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar); however, recent attempts by others failed to confirm these findings (15.Demarest K.T. Minor L.K. Gunnet J.W. J. Bone Miner. Res. 1997; 12 Suppl. 1: S413Google Scholar, 16.Pi M. Hinson T.K. Quarles L.D. J. Bone Miner. Res. 1999; 14: 1310-1319Crossref PubMed Scopus (63) Google Scholar, 17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 18.Bapty B.W. Dai L.J. Ritchie G. Jirik F. Canaff L. Hendy G.N. Quamme G.A. Kidney Int. 1998; 53: 583-592Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 19.Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar). Since cells may reduce CasR expression in culture, it remains unclear whether nontransformed osteoblasts or native osteoblasts express the known CasR. The detection of a functional CasR in bone cells derived from the mouse is potentially confounded by incomplete knowledge of the mouseCasR coding sequence (11.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar, 20.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar, 21.Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (98) Google Scholar) and the possible presence of another cation-sensing receptor in osteoblasts (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 20.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar). Prior studies by us (20.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar) and others (11.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar, 21.Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (98) Google Scholar) have identified only partial segments totaling 1514 bp of the mouse CasR cDNA sequence. Low abundance of CasR as well as nonspecificity of the detection methods may also contribute to the contradictory findings of CasR in osteoblasts (16.Pi M. Hinson T.K. Quarles L.D. J. Bone Miner. Res. 1999; 14: 1310-1319Crossref PubMed Scopus (63) Google Scholar). Whereas the variable reports of CasR expression in osteoblasts remain unexplained, all studies to date suggest that osteoblasts display a functional response to extracellular calcium and other cations via a G-protein-coupled receptor-like mechanism that resembles CasR (14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar, 15.Demarest K.T. Minor L.K. Gunnet J.W. J. Bone Miner. Res. 1997; 12 Suppl. 1: S413Google Scholar, 17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 19.Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar, 22.Kanatani M. Sugimoto T. Fukase M. Fujita T. Biochem. Biophys. Res. Commun. 1991; 181: 1425-1430Crossref PubMed Google Scholar, 23.Hartle J.E., II Pripic V. Siddhanti S.R. Spurney R.F. Quarles L.D. J. Bone Miner. Res. 1996; 11: 789-799Crossref PubMed Scopus (40) Google Scholar, 24.Sugimoto T.J. Kanatani M. Kano J. Tsukamoto H. Yamaguchi T. Fukase M. Chihara K. J. Bone Miner. Res. 1993; 12: 1445-1452Google Scholar, 25.Kanatani M. Sugimoto T. Fukase M. Fujita T. Biochem. Biophys. Res. Commun. 1991; 181: 1425-1430Crossref PubMed Scopus (74) Google Scholar, 26.Quarles L.D. Wenstrup R.J. Castillo S.A. Drezner M.K. Endocrinology. 1991; 128: 3144-3151Crossref PubMed Scopus (48) Google Scholar, 27.Quarles L.D. Hartle J.E., II Middleton J.P. Zhang J. Arthur J.M. Raymond J.R. J. Cell. Biochem. 1994; 56: 106-117Crossref PubMed Scopus (90) Google Scholar). Both DNA synthesis and chemotaxis are stimulated in osteoblasts by a panel of cations,viz. calcium, gadolinium, and neomycin (14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar, 17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 25.Kanatani M. Sugimoto T. Fukase M. Fujita T. Biochem. Biophys. Res. Commun. 1991; 181: 1425-1430Crossref PubMed Scopus (74) Google Scholar), that act on osteoblasts with relative potencies, apparent affinities, and specificities similar to their activation of CasR (14.Yamaguchi T. Chattopadhyay N. Kifor O. Butters R.R. Sugimoto T. Brown E.M. J. Bone Miner. Res. 1998; 13: 1530-1538Crossref PubMed Scopus (216) Google Scholar, 17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar,25.Kanatani M. Sugimoto T. Fukase M. Fujita T. Biochem. Biophys. Res. Commun. 1991; 181: 1425-1430Crossref PubMed Scopus (74) Google Scholar). Cations also induce c-fos mRNA expression and SRE-luciferase promoter activity in osteoblasts (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 26.Quarles L.D. Wenstrup R.J. Castillo S.A. Drezner M.K. Endocrinology. 1991; 128: 3144-3151Crossref PubMed Scopus (48) Google Scholar). The osteoblastic cation response is coupled to G-proteins as evidenced by [32P]GTP azidoanilide labeling of 42- and 38-kDa Gα-proteins in osteoblast membranes following cation stimulation (27.Quarles L.D. Hartle J.E., II Middleton J.P. Zhang J. Arthur J.M. Raymond J.R. J. Cell. Biochem. 1994; 56: 106-117Crossref PubMed Scopus (90) Google Scholar). In addition, the osteoblastic cation-sensing receptor inhibits agonist-induced stimulation of cAMP accumulation as reported forCasR (23.Hartle J.E., II Pripic V. Siddhanti S.R. Spurney R.F. Quarles L.D. J. Bone Miner. Res. 1996; 11: 789-799Crossref PubMed Scopus (40) Google Scholar). The functional properties of the putative osteoblasticCasR-like receptor differ, however, from those ofCasR in several ways. The cation specificities for the putative osteoblastic receptor, while overlapping, are not identical to those for CasR (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 19.Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar, 24.Sugimoto T.J. Kanatani M. Kano J. Tsukamoto H. Yamaguchi T. Fukase M. Chihara K. J. Bone Miner. Res. 1993; 12: 1445-1452Google Scholar, 28.Spurney R.F. Pi M. Flannery P. Quarles L.D. Kidney Int. 1999; 55: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). For example, the trivalent cation aluminum, which acts by a mechanism in common with gadolinium (26.Quarles L.D. Wenstrup R.J. Castillo S.A. Drezner M.K. Endocrinology. 1991; 128: 3144-3151Crossref PubMed Scopus (48) Google Scholar, 27.Quarles L.D. Hartle J.E., II Middleton J.P. Zhang J. Arthur J.M. Raymond J.R. J. Cell. Biochem. 1994; 56: 106-117Crossref PubMed Scopus (90) Google Scholar), is a potent agonist for the putative osteoblastic cation-sensing receptor, but not for CasR (28.Spurney R.F. Pi M. Flannery P. Quarles L.D. Kidney Int. 1999; 55: 1750-1758Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), whereas neither the CasR agonist magnesium (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar, 24.Sugimoto T.J. Kanatani M. Kano J. Tsukamoto H. Yamaguchi T. Fukase M. Chihara K. J. Bone Miner. Res. 1993; 12: 1445-1452Google Scholar) nor calcimimetics activate the putative cation receptor in osteoblasts (19.Mailland M. Waelchli R. Ruat M. Boddeke H.G. Seuwen K. Endocrinology. 1997; 138: 3601-3605Crossref PubMed Scopus (87) Google Scholar). Also, unlike CasR, the osteoblastic receptor does not appear to be coupled to phosphoinositide-specific phospholipase C. Rather, the effects of cations in osteoblasts are mediated by the activation of protein kinase C through unknown signaling pathways (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar). These functional differences suggest that a related but molecularly distinct calcium-sensing receptor may be present in osteoblasts (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar) and account for the inconsistent findings of CasR expression in osteoblasts. In an effort to prove the existence of another calcium-sensing receptor, we isolated and cloned the full-length mouse CasRcoding sequence and tested for CasR transcripts in osteoblasts using RT-PCR analysis with mouse CasR-specific primers. In addition, we evaluated the ability of a mouse anti-CasR antibody to detect CasR protein in osteoblasts by Western blot analysis. Finally, we determined whether the extracellular cation-sensing response is abolished in osteoblasts derived from mice deficient in CasR. Consistent with the presence of a novel osteoblastic cation-sensing mechanism, we found that cations stimulated to the same degree all measures of cation responses in osteoblasts derived from both wild-type andCasR-deficient mice. We used RT-PCR to isolate the full-length mouseCasR cDNA coding sequence. Total RNAs were prepared from mouse kidney tissue using the TRIzol reagent (Life Technologies, Inc.) as described previously (17.Quarles L.D. Hartle J.E. Siddhanti S.R. Guo R. Hinson T.K. J. Bone Miner. Res. 1997; 12: 393-402Crossref PubMed Scopus (103) Google Scholar). We designed mouseCasR-specific oligonucleotide primers based on the incompletely characterized mouse CasR sequence (11.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar, 20.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar, 21.Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (98) Google Scholar) as well as additional unpublished sequence information found in GenBankTM (accession number AF068900). For segments in which the mouse sequence was unknown, we substituted primers derived from regions of the rat CasR cDNA sequence that were highly conserved across species (2.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar). Using this approach, we were able to design six primer sets (see Table I) to overlapping segments covering the entire mouse cDNA open reading frame as well as portions of the 5′- and 3′-untranslated regions. RT-PCR was done using the TitanTM One Tube RT-PCR kit purchased from Roche Molecular Biochemicals. The reverse transcription reaction using 2.0 μg of total RNA treated with DNase I (Stratagene, La Jolla, CA) was incubated at 45 °C for 60 min, and the template was denatured at 94 °C for 2 min. PCR was performed with thermal cycling parameters of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 45 s for 10 cycles. This was followed by an additional 25 cycles with thermal cycling parameters of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 45 s plus an additional 5 s with each cycle. The reaction was completed with a final extension at 68 °C for 7 min. The resultant PCR products were subcloned into pCRII (Invitrogen, San Diego, CA) and sequenced using M13 forward and reverse primers. Sequence analysis was performed using the GCG software package (Version 8, Genetics Computer Group, Madison, WI).Table IOligonucleotide primers used for RT-PCR amplification of mouse CasR from kidneyNamePrimer sequencePositionaThe numbers refer to the nucleotide position within the mouse kidney calcium-sensing receptor cDNA sequence where the 5′-end of the primer will anneal.GenBank™Ref.1Facatg atgtc acttc tcagg1U103542.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar1Rtggag acggt gttac aggtg53721.Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (98) Google Scholar2Fcagcg agccc aaaag aaagg29121.Emanuel R.L. Adler G.K. Kifor O. Quinn S.J. Fuller F. Krapcho K. Brown E.M. Mol. Endocrinol. 1996; 10: 555-565Crossref PubMed Scopus (98) Google Scholar2Rcttca gaccg aaccc aatgg1190AF09294111.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar3Fcattg tcgtt ttctc cagcg1019AF0929413Racact caaaa cagca ggtgg1916AF06890011.Yamaguchi T. Chattopadhyay N. Kifor O. Brown E.M. Endocrinology. 1998; 139: 3561-3568Crossref PubMed Scopus (92) Google Scholar4Fggaag atctt gtgga gtggg1789AF0689004Rgcaaa gaaga agcag atggc2593AF00201520.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar5Ftcatc tgcat catct ggctc2431AF06890020.Hinson T. Damodaran T.V. Chen J. Zhang X. Qumsiyeh M.B. Seldin M.F. Quarles L.D. Genomics. 1997; 45: 279-289Crossref PubMed Scopus (27) Google Scholar5Racctt ctgtt tgcat ctcgg3121U103542.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar6Fcaaaa gcaac agcga agacc2969U103542.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar6Ractga gtaca ggctt tgacg3569U103542.Riccardi D. Lee W.S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholara The numbers refer to the nucleotide position within the mouse kidney calcium-sensing receptor cDNA sequence where the 5′-end of the primer will anneal. Open table in a new tab HeterozygousCasR knockout mice (CasR −/+) were obtained form the laboratory of Dr. David Conner (Harvard University, Boston, MA) (6.Ho C. Conner D.A. Pollak M.R. Ladd D.J. Kifor O. Warren H.B. Brown E.M. Seidman J.G. Seidman C.E. Nat. Genet. 1995; 11: 389-394Crossref PubMed Scopus (523) Google Scholar), and C57BL/6J mice expressing the large T antigen of SV40 were purchased from Jackson Laboratories (Bar Harbor, Maine). Mice were maintained and used in accordance with recommendations as described (37.National Research Council Guide for the Care and Use of Laboratory Animals.in: DHHS Publication NIH 86–23. Institute on Laboratory Animal Resources, Rockville, MD1985Google Scholar) and following guidelines established by the Institutional Animal Care and Use Committee of Duke University. Heterozygous CasR knockout females (CasR −/+) were mated with males expressing the large T antigen of SV40. The resulting male and female offspring that were heterozygous for expression of both the large T antigen of SV40 and CasR knockout were mated. Offspring of this mating were genotyped, and those that expressed the large T antigen of SV40 and either CasR +/+ (wild type) orCasR −/− (homozygous) were selected for isolation of calvarial osteoblasts. We used modifications of a nonenzymatic method for obtaining the osteoblastic cell lines (31.Ecarot-Charrier B. Glorieux F.H. van der Rest M. Pereira G. J. Cell Biol. 1983; 96: 639-643Crossref PubMed Scopus (296) Google Scholar). A fragment of the frontal and/or parietal bone from a single calvaria was aseptically removed from a 3–7-day-old mouse. Suture lines and endosteum were dissected away, and the bone fragment was placed in a culture dish. One or two metal strips were positioned on the endocranial surface and incubated for 3–4 days in Dulbecco's modified Eagle's medium/nutrient mixture F-12 containing 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin until the outgrowth of osteoblasts. The metal strips were removed, and the cells were allowed to grow until ∼60% confluent. The cells were subcultured and propagated by incubation in α-modified essential medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 50 μg/ml ascorbic acid in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. In addition, we also used established MC3T3-E1 (29.Quarles L.D. Yohay D.A. Lever L.W. Caton R. Wenstrup R.J. J. Bone Miner. Res. 1992; 7: 683-692Crossref PubMed Scopus (817) Google Scholar) and clonal TMOb-c (30.Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar) osteoblastic cell lines. These cells were grown in α-modified essential medium supplemented as described above in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were subcultured every 3–5 days using 0.001% Pronase. We used a PCR approach to detect wild-typeCasR +/+ and CasR −/−knockout osteoblasts expressing the large T antigen of SV40. Genomic DNA tissue was extracted and purified from the tail of each mouse or osteoblastic cell culture using a QIAamp blood kit (QIAGEN Inc., Valencia, CA). To detect the presence ofCasR −/−, we used the reverse primerCasR2144.R (5′-TGAAGCACCTACGGCACCTG-3′), specific for the native mouse CasR gene sequence, in combination with a primer designed for upstream elements in exon 4,CasR1956.F (5′-TGATGAAGAGTCTTTCTCGG-3′), or primer KCM-F (5′-TCTTGATTCCCACTTTGTGGTTCTA-3′) for the inserted neomycin gene sequence used for targeted disruption of exon 4 (32.Kovacs C.S. Ho-Pao C.L. Hunzelman J.L. Lanske B. Fox J. Seidman J.G. Seidman C.E. Kronenberg H.M. J. Clin. Invest. 1998; 101: 2812-2820Crossref PubMed Scopus (128) Google Scholar). Progeny containing the SV40 transgene were identified by PCR amplification of an ∼500-bp product of SV40 from individual genomic DNA using forward primer 5′-CAGAGCAGAATTGTGGAGTGG-3′ and reverse primer 5′-GGACAAACCACAACTAGAATGCAGTG-3′. PCR was performed with thermal cycling parameters of 94 °C for 3 min, 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 45 s for 35 cycles followed by a final extension at 72 °C for 10 min. Amplification products were resolved by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. To detect CasR expression, RT-PCR was done using two-step RNA PCR (Perkin-Elmer). In separate reactions, 2.0 μg of DNase-treated total RNA was reverse-transcribed into cDNA with the respective reverse primers specified below and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Reactions were carried out at 42 °C for 60 min followed by 94 °C for 5 min and 5 °C for 5 min. The products of first strand cDNA synthesis were directly amplified by PCR using AmpliTaq DNA polymerase (Perkin-Elmer) using three separate sets of primers based on the mouse CasR cDNA sequence. PCR was performed with thermal cycling parameters of 94 °C for 3 min, 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min for 30 cycles followed by a final extension at 72 °C for 10 min. The primer sets used to amplify overlapping regions of the 5′-end of mouse CasR included the mouse specific forward primer mCasR216.F (5′-AGAGCCATGGCATGGTTTGG-3′) and reverse primer mCasR653.R (5′-TGCTCCCACCACTGCGATGGTTG-3′) and the intron-spanning primer set consisting of the forward primer mCasR291.F (5′-CAGCGAGCCCAAAAG AAAGG-3′) and reverse primer mCasR1190.R (5′-CTTCA GACCG AACCC AATGG-3′). We documented that these primer sets were intron-spanning by comparing the sizes of the products derived from PCR of reverse-transcribed kidney RNA and genomic DNA. To amplify a region containing the 3′-end of CasR, we used the forward primer mCasR2431.F (5′-TCATC TGCAT CATCT GGCTC-3′ and the mouse specific reverse primer mCasR3537.R (5′-TTGGCTTCCTTGGGAAGACC-3′), located within the same exon. In addition, using a similar protocol (30.Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar), the mouse osteocalcin transcript was RT-PCR-amplified using the forward primer mOG+8.F (5′-CAAGTCCCACACAGCAGCTT-3′) and the reverse primer mOG+378.R (5′-AAAGCCGAGCTGCCAGAGTT-3′). Mouse β-actin was amplified as a control for the RT-PCRs as described previously (30.Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar). Amplification products were resolved by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. We analyzed alkaline phosphatase in cell layers by colorimetric assay of enzyme activity with the substrate p-nitrophenol phosphate as described previously (26.Quarles L.D. Wenstrup R.J. Castillo S.A. Drezn
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