MEPE-ASARM Peptides Control Extracellular Matrix Mineralization by Binding to Hydroxyapatite: An Inhibition Regulated by PHEX Cleavage of ASARM

Addison, William N.; Nakano, Yukiko; Loisel, Thomas P.; Crine, Phillippe; McKee, Marc D.

doi:10.1359/jbmr.080601

Cited by 177 publications

(231 citation statements)

References 53 publications

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“…However, a recent report (37) also suggests that PHEX degrades the circulating phospho-ASARM peptide, which inhibits matrix mineralization. During normal matrix mineralization in vitro, treatment of MC3T3-E1 cells with exogenous PHEX alone does not result in a significant increase in mineralization and PHEX rescues mineralization inhibition when exogenous phospho-ASARM is present (37). Collectively, we contend that when the PHEX level is greater than the MEPE level, MEPE is protected from cathepsin B cleavage action by PHEX.…”

Section: Mepe Expression Is Strongly Stimulated By Bmp-2 Treatment Anmentioning

confidence: 74%

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Cho¹,

Yoon²,

Woo³

et al. 2009

Journal of Biological Chemistry

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Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knockdown experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.Mineral homeostasis in the body is critical for healthy bones and teeth, and is generally regulated by the calcium-phosphate balance in the bone and kidney networks. In the past, the vitamin D/parathyroid hormone axis was assumed to be a single major circuit in bone-renal phosphate regulation, but recently, new bone-renal phosphate regulating factors have been identified. The finding of fibroblast growth factor 23 (FGF23), 2 phosphate-regulating genes with homologies to endopeptidases on the X chromosome (PHEX) and matrix extracellular phosphoglycoprotein (MEPE) genes, and their pathophysiological roles in the genetic diseases of mineral metabolism, have provided a great deal of insight into the understanding of bone-and mineral-related diseases. Among these, autosomal-dominant hypophosphatemic rickets (OMIM number 193100) is characterized by renal phosphate wasting, hypophosphatemia, and inappropriately normal 1,25-dihydroxyvitamin D 3 levels (1). Autosomal-dominant hypophosphatemic rickets is caused by a missense mutation of FGF23, which is resistant to proteolysis by PHEX and increases the half-life of full-length phosphaturic FGF23 (2). Second, X-linked hypophosphatemic...

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Section: Mepe Expression Is Strongly Stimulated By Bmp-2 Treatment Anmentioning

confidence: 74%

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Cho¹,

Yoon²,

Woo³

et al. 2009

Journal of Biological Chemistry

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“…(10,14,67) The ASARM region in human MEPE spans residues 508 to 525 at the C-terminal end of the protein, whereas in OPN it is found more centrally. (13,14,68) Previously, we demonstrated that PHEX is able to cleave the potent mineralization-inhibiting ASARM peptide from MEPE and OPN. (13,14) In addition, we reported on ASARM binding to hydroxyapatite mineral to provide a mechanistic explanation for how loss of PHEX activity in XLH might lead to extracellular matrix accumulation of ASARM, resulting in osteomalacia.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, previous in vivo studies have shown partial correction of the osteomalacic phenotype when Phex/PHEX is overexpressed in osteoblasts, (83,84) and local, microenvironmental effects of PHEX within the extracellular matrix have been explicitly proposed. (13,14,48) The mouse bone extracts used in this study derive from the demineralized fraction enriched in the mineral-binding peptides/proteins thought to be regulating matrix mineralization. We demonstrate by immunoblotting the accumulation of a $35-kDa OPN fragment in extracts of Hyp mouse bone that is absent in bone extracts from wild-type counterpart mice.…”

Section: Discussionmentioning

confidence: 99%

“…As part of this process, enzymatic activity may be further controlled by enzymeinhibiting interactions, such as that observed between PHEX and glycosaminoglycans, (36) or by temporal expression of PHEX. (53,62) Given the potent mineralization-inhibiting activity of OPN and its phosphorylated peptide motifs such as the ASARM region, (13,14) it is reasonable to consider the notion that the required, extensive/complete degradation of OPN by PHEX does not occur in XLH, thus contributing to the osteomalacic phenotype.…”

Section: Discussionmentioning

confidence: 99%

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Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

Barros

Hoac

Neves

et al. 2012

Journal of Bone and Mineral Research

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111

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X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine-and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an $35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. ß

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“…MEPE proteolysis generates the protease-resistant acidic serine aspartate rich MEPE associated motif (ASARM) peptide, which directly inwhich directly inhibits calcium oxalate crystallization and crystal growth on the extracellular matrix (Hoyer et al 2001) and promotes inhibition of the renal NPT2 phosphate co-trans- Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts porter, affecting renal phosphate handling (Rowe et al 2004). The MEPE-derived ASARM peptide must be phosphorylated to play its role as a PHEX substrate (Addison et al 2008). It was recently demonstrated that osteopontin (OPN), a member of the small integrin-binding ligand N-linked glycoproteins family, is also a source of ASARM peptide.…”

mentioning

confidence: 99%

Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

Silva

Tempone

Silva

et al. 2010

Mem. Inst. Oswaldo Cruz

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MEPE-ASARM Peptides Control Extracellular Matrix Mineralization by Binding to Hydroxyapatite: An Inhibition Regulated by PHEX Cleavage of ASARM

Cited by 177 publications

References 53 publications

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

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