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• W1994303022 abstract "Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex inHyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalizedPhex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phextransgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phexfunction in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect thePhex substrate FGF-23 in osteoblasts. Taken together, thesein vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites ofPhex expression and/or additional factors are likely to be important in the pathogenesis of XLH. Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex inHyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalizedPhex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phextransgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phexfunction in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect thePhex substrate FGF-23 in osteoblasts. Taken together, thesein vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites ofPhex expression and/or additional factors are likely to be important in the pathogenesis of XLH. X-linked hypophosphatemia reverse transcription internal ribosome entry site green fluorescent protein reverse transcriptase-PCR benzyloxycarbonyl–Ala-Ala-Leu-p-nitroanilide 4-morpholineethanesulfonic acid bone mineral density Because the discovery that mutations ofPHEX, or the Phosphate-regulating gene with homologies to Endopeptidases on the Xchromosome, is the genetic defect underlying X-linked hypophosphatemia (XLH)1 (1Anonymous Nat. Genet. 1995; 11: 130-136Crossref PubMed Scopus (957) Google Scholar, 2Strom T.M. Francis F. Lorenz B. Boddrich A. Econs M.J. Lehrach H. Meitinger T. Hum. Mol. Genet. 1997; 6: 165-171Crossref PubMed Scopus (174) Google Scholar, 3Grieff M. Mumm S. Waeltz P. Mazzarella R. Whyte M.P. Thakkar R.V. Schlessinger D. Biochem. Biophys. Res. Commun. 1997; 231: 635-639Crossref PubMed Scopus (50) Google Scholar, 4Holm I.A. Huang X. Kunkel L.M. Am. J. Hum. Genet. 1997; 60: 790-797PubMed Google Scholar), efforts have been underway to determine how this novel endopeptidase regulates phosphorus and mineral homeostasis. Phex is one of six members of the M13 family of zinc-dependent type II cell-surface membrane metalloproteases (5Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 6Lee S. Zambas E.D. Marsh W.L. Redman C.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6353-6357Crossref PubMed Scopus (202) Google Scholar, 7Kiryu-Seo S. Sasaki M. Yokohama H. Nakagomi S. Hirayama T. Aoki S. Wada K. Kiyama H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4345-4350Crossref PubMed Scopus (102) Google Scholar). The presence of renal phosphate wasting secondary to inactivating mutations of thePhex gene suggests that this endopeptidase degrades a novel phosphaturic hormone (referred to as phosphatonin) or inactivates a phosphate-conserving factor (8Quarles L.D. Drezner M.K. J. Clin. Endocrinol. & Metab. 2001; 86: 494-496Crossref PubMed Scopus (0) Google Scholar). Neutral endopeptidase substrates ZAAL-pNA and [Leu]enkephalin, as well as certain parathyroid hormone-related peptides (9Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. & Metab. 2001; 281: 837-847Crossref PubMed Google Scholar, 10Lipman M.L. Dibyendu P. Hugh P.J. Bennett J.E. Henderson E.S. Yingnian S. Goltzman D. Karaplis A.C. J. Biol. Chem. 1998; 273: 13729-13737Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Shirotani K. Tsubuki S. Iwata N. Takaki Y. Harigaya W. Maruyama K. Kiryu-Seo S. Kiyama H. Iwata H. Tomita T. Iwatsubo T. Saido T.C. J. Biol. Chem. 2001; 276: 21895-21901Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 12Boileau G. Tenenhouse H.S. Desgroseillers L. Crine P. Biochem. J. 2001; 355: 707-713Crossref PubMed Scopus (72) Google Scholar), are cleaved by recombinant Phex in vitro, but the biological relevance of these substrates is not certain. Recent studies indicate that FGF-23 may be a physiologically important Phex substrate and a candidate for phosphatonin (14Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (69) Google Scholar). Not only is FGF-23 cleaved by recombinant Phex in vitro (13Bowe A.E. Finnegan R. Jan de Beur S.M. Cho J. Levine M.A. Kumar R. Schaivi S.C. Biochem. Biophys. Res. Commun. 2001; 284: 977-981Crossref PubMed Scopus (294) Google Scholar), but mutations in the FGF-23 gene product cause the related disorder autosomal dominant hypophosphatemia (14Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (69) Google Scholar), and FGF-23 induces hypophosphatemia and defective mineralization when administered to mice in vivo(15Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1193) Google Scholar).There is significant evidence indicating bone is a physiologically relevant site of Phex expression and is directly involved in the pathogenesis of XLH. Phex is expressed at high levels in osteoblasts and other mineralizing tissues such as teeth and growth plate cartilage (16Beck L. Soumounou Y. Martel J. Krishnamurthy G. Gauthier C. Goodyer C.G. Tenenhouse H.S. J. Clin. Invest. 1997; 99: 1200-1209Crossref PubMed Scopus (248) Google Scholar, 17Du L. Debarats M. Viel J. Glorieux F.H. Cawthorn C. Ecarot B. Genomics. 1996; 36: 22-28Crossref PubMed Scopus (166) Google Scholar, 18Guo R. Quarles L.D. J. Bone Miner. Res. 1997; 12: 1009-1017Crossref PubMed Scopus (103) Google Scholar, 19Zoidis E. Zapf J. Schmid C. Mol. Cell. Endocrinol. 2000; 168: 41-51Crossref PubMed Scopus (29) Google Scholar, 20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar) where its expression is temporally associated with the formation of mineralized extracellular matrix in cultured osteoblasts (18Guo R. Quarles L.D. J. Bone Miner. Res. 1997; 12: 1009-1017Crossref PubMed Scopus (103) Google Scholar, 20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar). In addition, available data (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar, 22Delvin E.E. Richard P. Desbarats M. Ecarot-Charrier B. Glorieux F.H. Bone. 1990; 11: 87-94Crossref PubMed Scopus (11) Google Scholar) suggest that loss of Phex function in osteoblasts results in a nascent defect that leads to impaired mineralization of extracellular matrix, independent of the hypophosphatemia. Osteoblasts derived fromHyp mice, a murine homologue of XLH, display defective mineralization and other abnormalities in culture (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar, 22Delvin E.E. Richard P. Desbarats M. Ecarot-Charrier B. Glorieux F.H. Bone. 1990; 11: 87-94Crossref PubMed Scopus (11) Google Scholar), as well as fail to form mineralized bone after transplantation into normal mice (23Ecarot B. Glorieux F.H. Desbarats M. Travers R. Labelle L. J. Bone Miner. Res. 1992; 7: 523-530Crossref PubMed Scopus (46) Google Scholar). Finally, putative phosphate and mineralization inhibitory activities also have been identified in conditioned media ofHyp osteoblasts (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar, 24Nesbitt T. Fujiwara I. Thomas R. Xiao Z.S. Quarles L.D. Drezner M.K. J. Bone Miner. Res. 1999; 14: 2027-2035Crossref PubMed Scopus (48) Google Scholar), suggesting that Phexand its substrate are both produced in bone.Although these data provide compelling evidence that the loss ofPhex function in osteoblasts is causally related to the intrinsic abnormality of mineralization, several components of this model have not be substantiated. First, there is no direct evidence that Phex metabolizes endogenous phosphaturic or mineralization inhibitory factors synthesized by osteoblasts. Second, FGF-23, the current best candidate for phosphatonin (14Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (69) Google Scholar, 15Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1193) Google Scholar), has not been shown to be expressed in bone marrow (14Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (69) Google Scholar), although its expression in osteoblasts has not been excluded. Third, several studies (25Rifas L. Cheng S.L. Halstead L. Gupta A. Husker K.A. Avail L. Calcify. Tissue Int. 1997; 61: 256-259Crossref PubMed Scopus (26) Google Scholar, 26Gundberg C. Clough M.E. Carpenter T.O. Endocrinology. 1992; 130: 1909-1915Crossref PubMed Scopus (41) Google Scholar) have failed to document nascent defects in Hyp-derived osteoblasts, indicating that the observed abnormalities of cultured osteoblasts may be secondary to the Hyp milieu. Fourth, the observation that parabiosis (27Meyer R.A. Meyer M.H. Gray R.W. J. Bone Miner. Res. 1989; 4: 493-500Crossref PubMed Scopus (192) Google Scholar) and cross-kidney transplantation (28Lajeunesse D. Meyer R.A. Hamel L. Kidney Int. 1996; 50: 1531-1538Abstract Full Text PDF PubMed Scopus (70) Google Scholar) between normal and Hyp mice lead to phosphaturia in the normal animal indicates the presence of extrinsic circulating factors (29Nesbitt T. Coffman T.M. Griffiths R. Drezner M.K. J. Clin. Invest. 1992; 89: 1453-1459Crossref PubMed Scopus (207) Google Scholar) that modulate bone mineralization in the X-linked disorder, either directly or indirectly through the induction of hypophosphatemia. Finally, bone marrow transplantation via the intraperitoneal route, which does not normalize Phex expression in bone, partially rescues the hypophosphatemia in Hyp mice (30Miyamura T. Tanaka H. Inoue M. Ichinose Y. Seino Y. J. Bone Miner. Res. 2000; 15: 1451-1458Crossref PubMed Scopus (25) Google Scholar). All of these studies indicate that factors extrinsic to the osteoblast might be responsible for the mineralization defect in XLH. No studies to date, however, have examined whether the apparent intrinsic abnormalities of mineralization in Hyp-derived osteoblasts are corrected by restoration of Phex expression and function in osteoblasts.In the present investigation, we directly examined whetherPhex deficiency is directly associated with impairment of osteoblast-mediated mineralization by two complementary approaches. We used a retroviral vector to overexpress a functional PhexcDNA in osteoblasts derived from Hyp mice and the osteocalcin (OG2) promoter to achieve targeted overexpression of Phex to Hyp osteoblastsin vivo. Neither of these approaches corrected theHyp-related mineralization abnormalities, and the targeted expression of Phex to mature osteoblasts in bone did not rescue the hypophosphatemia. Thus, our findings fail to support the simple hypothesis that loss of Phex in osteoblasts is primarily responsible for the Hyp phenotype.DISCUSSIONBecause Phex is predominantly expressed in bone and osteoblasts derived from Hyp mice have an apparent intrinsic mineralization defect, we (8Quarles L.D. Drezner M.K. J. Clin. Endocrinol. & Metab. 2001; 86: 494-496Crossref PubMed Scopus (0) Google Scholar) and others (16Beck L. Soumounou Y. Martel J. Krishnamurthy G. Gauthier C. Goodyer C.G. Tenenhouse H.S. J. Clin. Invest. 1997; 99: 1200-1209Crossref PubMed Scopus (248) Google Scholar, 17Du L. Debarats M. Viel J. Glorieux F.H. Cawthorn C. Ecarot B. Genomics. 1996; 36: 22-28Crossref PubMed Scopus (166) Google Scholar, 20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar) favored the hypothesis that diminished Phex expression in osteoblasts was primarily responsible for the pathogenesis of the XLH and theHyp phenotype. Direct evidence that co-expression ofPhex and its putative substrates in the bone milieu participated in bone mineralization and regulation of systemic phosphate homeostasis, however, was lacking (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar, 24Nesbitt T. Fujiwara I. Thomas R. Xiao Z.S. Quarles L.D. Drezner M.K. J. Bone Miner. Res. 1999; 14: 2027-2035Crossref PubMed Scopus (48) Google Scholar). The results from the current study indicate that Phex expression in osteoblasts is not sufficient to explain the pathogenesis of XLH. Rather, we found that the targeted overexpression of Phex to osteoblasts in Hyp mice using the osteocalcin promoter failed to correct either the mineralization defect of bone or the systemic hypophosphatemia (Figs. Figure 6, Figure 7, Figure 8). Despite attaining expression ofPhex in mature osteoblasts to levels (Fig. 8b) and activity (Fig. 6e) comparable with endogenousPhex, OG2-Phex-Hyp mice exhibited hypophosphatemia (Fig. 7a), radiographic evidence of rickets (Fig. 8a), and histologic evidence of osteomalacia (Fig.8c) identical to that of non-transgenic Hyplittermates. The overexpression of Phex also had no demonstrable effect in normal OG2-Phex mice, which displayed serum phosphate levels (Fig. 7a) and skeletal morphology (Fig. 8) indistinguishable from non-transgenic littermates, despite higher endopeptidase activity against a syntheticPhex substrate (Fig. 6e). Thus, our findings fail to support the hypothesis that abnormal Phex function in mature osteoblasts plays a primary role in the pathogenesis of hypophosphatemia and raises questions regarding whether Phexhas a direct or indirect role in regulating osteoblast-mediated mineralization of bone.Based on our findings, it is likely that expression of Phexat sites other than those controlled by the osteocalcin promoter is responsible for the persistent accumulation of Phexsubstrates, resulting in hypophosphatemia and the failure to rescue theHyp phenotype. Although Phex and osteocalcin expression are concordant in mature osteoblasts and osteocytes (20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar), the osteocalcin promoter does not express Phex in teeth, cartilage, or other sites where Phex is normally present (20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar) and initiates expression later during embryogenesis at embryonic day 15.5 compared to day 11 for endogenousPhex. 2S. Liu, R. Guo, Q. Tu, and L. D. Quarles, unpublished observations.Consequently, persistent abnormalities in the spatial and temporal expression of Phex, and/or the failure to restore the full complement of Phex activities in the whole mouse (which may be the sum of Phex expression in many tissues), may account for the failure to rescue the Hyp phenotype in the current studies. In addition, other genes and environmental factors have been shown to affect the severity of hypophosphatemic rickets (44Holm I.A. Nelson A.E. Robinson B.G. Mason R.S. Marsh D.J. Cowell C.T. Carpenter T.O. J. Clin. Endocrinol. & Metab. 2001; 86: 3889-3899Crossref PubMed Scopus (127) Google Scholar) and also could impact upon the inability of Phex to rescue theHyp phenotype.Other potential explanations for our in vivo findings seem unlikely. Even though recent reports indicate that mutantPhex proteins accumulate in the endoplasmic reticulum (45Sabbagh Y. Boileau G. DesGroseillers L. Tenenhouse H.S. Hum. Mol. Genet. 2001; 10: 1539-1546Crossref PubMed Scopus (48) Google Scholar), we have shown that the 3′ deletion Phex mutant does not interfere with the function of the wild-type transfectedPhex. For example, overexpression of the mutantPhex construct neither interferes with the mineralization of normal osteoblasts in vitro (Fig. 4) nor disrupts the enzymatic activity of wild-type Phex in vitro (9Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. & Metab. 2001; 281: 837-847Crossref PubMed Google Scholar). In addition, our experimental design, using the 1.3-kb OG2 promoter fragment to drive expression of transgenes restricted to mature osteoblasts in vivo (34Frendo J.L. Xiao G. Fuchs S. Franceschi R.T. Karsenty G. Ducy P. J. Biol. Chem. 1998; 273: 30509-30516Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), achieved the successful restoration of Phex endopeptidase activity in bone (Fig.6e). Demonstration of Phex activity in osteoblasts and calvaria also lessens the concern that endogenous Osteocalcin may inhibit the activity of co-expressed Phex, as suggested by a recent in vitro studies (12Boileau G. Tenenhouse H.S. Desgroseillers L. Crine P. Biochem. J. 2001; 355: 707-713Crossref PubMed Scopus (72) Google Scholar). Finally, because Phex is not expressed in osteoblastic precursors (20Ruchon A.F. Tenenhouse H.S. Marcinkiewicz M. Siegfried G. Aubin J.E. DesGroseillers L. Crine P. Boileau G. J. Bone Miner. Res. 2000; 15: 1440-1450Crossref PubMed Scopus (122) Google Scholar), it seems unlikely that targeting Phex to earlier states of the osteoblast lineage would have altered the results.We did observe a slight, but significant, increase in dry ashed weight and bone mineral density of femurs derived from OG2 Phex-Hyp mice (Fig. 7, b and c). These later findings, representing changes in bone mineralization not detected by conventional radiographic or histologic methods, raise the possibility that Phex may have a role in regulating bone mineralization that is masked by the persistent hypophosphatemia in theOG2 Phex-Hyp mice. It is possible thatPhex directly or indirectly regulates the production of matrix proteins and/or local bone substrates, which in turn regulate the mineralization process and account for the small increase in bone mineral density in persistently hypophosphatemicOG2-Phex-Hyp mice. Proof of an effect independent of hypophosphatemia mediated by Phex direct regulation of osteoblast-mediated mineralization requires identification of additional physiologically important Phex substrates in bone. Nevertheless, the observed increase in bone mass in OG2 Phex-Hyp mice is in keeping with the increased production of factors that regulate mineralization and phosphate transport by cultured osteoblasts derived from Hyp mice (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar,23Ecarot B. Glorieux F.H. Desbarats M. Travers R. Labelle L. J. Bone Miner. Res. 1992; 7: 523-530Crossref PubMed Scopus (46) Google Scholar). Recent studies have observed abnormalities of bone extracellular matrix proteins (46Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar) and the accumulation of MEPE (47Argiro L. Desbarats M. Glorieux F.H. Ecarot B. Genomics. 2001; 74: 342-351Crossref PubMed Scopus (142) Google Scholar) inHyp mice. The failure to detect FGF-23, the only known physiologic Phex substrate and the leading candidate for phosphatonin (13Bowe A.E. Finnegan R. Jan de Beur S.M. Cho J. Levine M.A. Kumar R. Schaivi S.C. Biochem. Biophys. Res. Commun. 2001; 284: 977-981Crossref PubMed Scopus (294) Google Scholar, 14Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (69) Google Scholar, 35Bonnerot C. Nicolas J.F. Methods Enzymol. 1993; 225: 451-469Crossref PubMed Scopus (91) Google Scholar), in bone (Fig. 5) and bone marrow (15Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1193) Google Scholar), however, indicates that this phosphaturic factor is not the putativePhex substrate in the local bone environment. Related members of the M13 family of metalloproteases have multiple substrates whose tissue-specific actions are derived from their co-localization (8Quarles L.D. Drezner M.K. J. Clin. Endocrinol. & Metab. 2001; 86: 494-496Crossref PubMed Scopus (0) Google Scholar). Therefore, Phex may separately metabolize distinct substrates that regulate phosphaturia and mineralization.We confirmed previous reports (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar) that osteoblasts derived fromHyp mice exhibit the inability to form mineralization nodules in culture (Figs. 3 and 4). However, restoration ofPhex expression (Fig. 1) and enzymatic activity (Fig. 2) toHyp osteoblasts did not restore their capacity to mineralize extracellular matrix in vitro (Fig. 3) under culture conditions supporting mineralization in normal osteoblasts (Fig. 4). The inability to rescue the Hyp osteoblastic phenotype byPhex overexpression in vitro is consistent with prior studies where co-culture of cells expressing Phex also failed to correct the mineralization defect in Hyposteoblasts (21Xiao 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 current negative findings are not due to the effects of the retroviral transduction or inadequate restoration of endopeptidase activity. Moreover, the presence of a truncatedPhex in Hyp osteoblast does not interfere with the restoration of Phex function, because overexpression of a 3′-truncated mutant Phex failed to disrupt mineralization of normal osteoblasts (Fig. 4). It also is unlikely that aberrant temporal and/or the excessive amounts of retroviral mediatedPhex expression or GFP could have influenced our resultsin vitro, because transduction of normal osteoblasts with the retroviral Phex construct with and without GFP did not affect their ability to mineralize (Fig. 4). We cannot exclude, however, potential variability resulting from differences in the cell culture models used to assess mineralization by alizarin red staining. Indeed, a preliminary report from another laboratory (48Sabbagh Y. Londowski J.M. Mathiesen D. Gauthier C. Boileau G. Tenenhouse H.S. Poeschla E.M. Kumar R. J. Am. Soc. Nephrol. 2000; 11: 413APubMed Google Scholar), unlike ourin vitro studies, shows that overexpression ofPhex in Hyp-derived osteoblasts, although not sufficient to fully normalize mineralization, results in partial rescue of their mineralization capacity.Regardless, the in vitro studies of Hyp-derived osteoblasts indicate a more complex pathogenesis of the defective mineralization in cultured osteoblasts. There are differences in the gene expression profiles in osteoblastic cultures compared with bone inHyp mice, indicating that these culture models do not fully mimic the in vivo state (21Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar, 46Miao D. Bai X. Panda D. McKee M. Karaplis A. Goltzman D. Endocrinology. 2001; 142: 926-939Crossref PubMed Scopus (139) Google Scholar). In addition, the exposure to hypophosphatemia or the Hyp milieu may somehow limit the expression of an accessory factor necessary for Phexfunction and/or lead to deficiencies in the full complement of genes necessary for mineralization. There is a precedent for hypophosphatemia to induce a similar intrinsic mineralization defect in osteoblasts derived from mice in which the renal sodium-dependent phosphate transporter has been ablated (49Thomas R. Fujiwara I. Tenenhouse H.S. Quarles L.D. Drezner M.K. J. Bone Miner. Res. 1999; 14: 189Crossref Scopus (21) Google Scholar). Extracellular phosphate also may alter osteoblast gene expression through its actions to modulate nuclear export of the osteoblast transcriptional regulatorCbfa1 in bone cells (33Fujita T. Izumo N. Fukuyama R. Meguro T. Nakamuta H. Kohno T. Koida M. Biochem. Biophys. Res. Commun. 2001; 280: 348-352Crossref PubMed Scopus (65) Google Scholar). Identification of the potentialPhex substrates in bone and these modulating factors will be necessary to unravel the relative contribution of local and systemic regulation of in osteoblast-mediated mineralization.In conclusion, our current findings fail to support the simple hypothesis that the lack of Phex in Hyposteoblasts is directly responsible for the impaired mineralization and abnormalities in systemic phosphate homeostasis. Rather, other sites and/or temporal aspects of Phex expression appear to be physiologically important in the metabolism of the phosphaturic factor phosphatonin. It is likely that the successful rescue of theHyp phenotype will require restoration of normalPhex activity in one or more of these additional sites. In addition, we failed to establish a cause and effect relationship between Phex expression in osteoblasts and their ability to form a mineralized extracellular matrix in culture, indicating that hypophosphatemia has a predominant role in the defective mineralization. Additional studies that restore endogenousPhex expression in transgenic animals (possibly by usingPhex promoter) and/or selectively disrupt Phex by tissue-specific targeted deletion strategies will be necessary to establish a cause and effect relationship between the site ofPhex expression and the Hyp phenotype. In addition, confirming the identify of phosphatonin, as well as the identification of possible bone-derived substrates for Phexand/or phosphate-dependent accessory factors that regulate the mineralization process, will be important in unraveling the complex pathogenesis of XLH. Because the discovery that mutations ofPHEX, or the Phosphate-regulating gene with homologies to Endopeptidases on the Xchromosome, is the genetic defect underlying X-linked hypophosphatemia (XLH)1 (1Anonymous Nat. Genet. 1995; 11: 130-136Crossref PubMed Scopus (957) Google Scholar, 2Strom T.M. Francis F. Lorenz B. Boddrich A. Econs M.J. Lehrach H. Meitinger T. Hum. Mol. Genet. 1997; 6: 165-171Crossref PubMed Scopus (174) Google Scholar, 3Grieff M. Mumm S. Waeltz P. Mazzarella R. Whyte M.P. Thakkar R.V. Schlessinger D. Biochem. Biophys. Res. Commun. 1997; 231: 635-639Crossref PubMed Scopus (50) Google Scholar, 4Holm I.A. Huang X. Kunkel L.M. Am. J. Hum. Genet. 1997; 60: 790-797PubMed Google Scholar), efforts have been underway to determine how this novel endopeptidase regulates phosphorus and mineral homeostasis. Phex is one of six members of the M13 family of zinc-dependent type II cell-surface membrane metalloproteases (5Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 6Lee S. Zambas E.D. Marsh W.L. Redman C.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6353-6357Crossref PubMed Scopus (202) Google Scholar, 7Kiryu-Seo S. Sasaki M. Yokohama H. Nakagomi S. Hirayama T. Aoki S. Wada K. Kiyama H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4345-4350Crossref PubMed Scopus (102) Google Scholar). The presence of renal phosphate wasting secondary to inactivating mutations of thePhex gene suggests that this endopeptidase degrades a novel phosphaturic hormone (referred to as phosphatonin) or inactivates a phosphate-c" @default.
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• W1994303022 title "Overexpression of Phex in Osteoblasts Fails to Rescue the Hyp Mouse Phenotype" @default.
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