Yukiko Nakano

MEPE-ASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: an inhibition regulated by PHEX cleavage of ASARM.

Hyp mice having an inactivating mutation of the phosphate‐regulating gene with homologies to endopeptidases on the X‐chromosome (Phex) gene have bones with increased matrix extracellular phosphoglycoprotein (MEPE). An acidic, serine‐ and aspartic acid–rich motif (ASARM) is located in the C terminus of MEPE and other mineralized tissue matrix proteins. We studied the effects of ASARM peptides on mineralization and how PHEX and MEPE interactions contribute to X‐linked hypophosphatemia (XLH). ASARM immunoreactivity was observed in the osteoid of wildtype bone and in the increased osteoid of Hyp mice. In wildtype bone, PHEX immunostaining was found particularly in osteoid osteocytes and their surrounding matrix. Treatment of MC3T3‐E1 osteoblasts with triphosphorylated (3 phosphoserines) ASARM peptide (pASARM) caused a dose‐dependent inhibition of mineralization. pASARM did not affect collagen deposition or osteoblast differentiation, suggesting that pASARM inhibits mineralization by direct binding to hydroxyapatite crystals. Binding of pASARM to mineralization foci in pASARM‐treated cultures and to synthetic hydroxyapatite crystals was confirmed by colloidal‐gold immunolabeling. Nonphosphorylated ASARM peptide showed little or no binding to hydroxyapatite and did not inhibit mineralization, showing the importance of ASARM phosphorylation in regulating mineralization. PHEX rescued the inhibition of osteoblast culture mineralization by pASARM, and mass spectrometry of cleaved peptides obtained after pASARM‐PHEX incubations identified pASARM as a substrate for PHEX. These results, showing that pASARM inhibits mineralization by binding to hydroxyapatite and that this inhibitor can be cleaved by PHEX, provide a mechanism explaining how loss of PHEX activity can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia of XLH.

Expression and localization of plasma transglutaminase factor XIIIA in bone.

Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immuno-histochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immuno-reactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.

Eccentric localization of osteocytes expressing enzymatic activities, protein, and mRNA signals for type 5 tartrate-resistant acid phosphatase (TRAP).

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap-positive osteocytes thus identified were confined to the areas no further than 200 üm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

Regulation of ATPase activity of transglutaminase 2 by MT1‐MMP: Implications for mineralization of MC3T3‐E1 osteoblast cultures

A pro‐mineralization function for transglutaminase 2 (TG2) has been suggested in numerous studies related to bone, cartilage, and vascular calcification. TG2 is an enzyme which can perform protein crosslinking functions, or act as a GTPase/ATPase depending upon different stimuli. We have previously demonstrated that TG2 can act as an ATPase in a Ca2+‐rich environment and that it can regulate phosphate levels in osteoblast cultures. In this study, we investigate the role MT1‐MMP in regulating the ATPase activity of TG2. We report that proteolytic cleavage of TG2 by MT1‐MMP in vitro results in nearly a 3‐fold increase in the ATPase activity of TG2 with a concomitant reduction in its protein‐crosslinking activity. We show that MC3T3‐E1 osteoblasts secreted full‐length TG2 and major smaller fragments of 66 and 56 kDa, the latter having ATP‐binding abilities. MT1‐MMP inhibition by a neutralizing antibody suppressed mineralization of osteoblast cultures to 35% of control, and significantly reduced phosphate levels in conditioned medium (CM). Furthermore, MT1‐MMP inhibition abolished two of TG2 fragments in the cultures, one of which, the 56‐kDa fragment, has ATPase activity. Neutralization of MT1‐MMP at early phases of mineralization significantly reduced mineral deposition, but had no effect in later phases implying MT1‐MMP and TG2 might contribute to the initiation of mineralization. The cleavage of TG2 by MT1‐MMP likely occurs on the cell surface/pericellular matrix where MT1‐MMP and TG2 were co‐localized. Based on these data, we propose that MT1‐MMP modulates the extracellular function TG2 as part of a regulatory mechanism activates the pro‐mineralization function of TG2. J. Cell. Physiol. 223: 260–269, 2010.

Expression of bone type 1 PTH receptor in rats with chronic renal failure

Some researchers have speculated that a decrease in bone type 1 PTH receptor (PTH1R) may be among the causes of “skeletal resistance” in chronic renal failure (CRF). Indeed, the down-regulation of PTH1R mRNA has been identified in uremic bones. However, few studies have identified the patterns of PTH1R protein expression. In this article we compare the bone expression of PTH1R protein and mRNA under control and CRF conditions. Sprague–Dawley rats underwent 5/6 nephrectomies (Nx) or sham operations (control), and were killed 16 weeks later. Blood urea nitrogen (BUN), serum Cr, P, and parathyroid hormone (PTH) were higher in the Nx group than in the controls, while serum Ca and 1,25(OH)2D3 were lower in the Nx group. Immunohistochemical images of lumbar bone samples were analyzed by an image processing system. PTH1R was essentially identified in all osteoblasts. The expression of osteoblast PTH1R protein was quantified based on the gray value of PTH1R staining. The mean gray scale of osteoblasts was 25% lower in Nx rats than in control rats (P < 0.01), whereas osteoblast cell counts and cell sizes were not significantly different between the two groups. Thus, down-regulation of PTH1R protein expression under the CRF condition appeared likely. Total RNA extracted from the bone samples was reverse transcribed for real-time polymerase chain reaction (PCR). PTH1R mRNA expression was 33% lower in the Nx group than in the control group in the quantitative PCR analysis (P < 0.05). Our findings suggested that osteoblast PTH1R expression is down-regulated at both the protein and mRNA levels in the steady state of CRF.

Alkaline and Acid Phosphatases in Bone Cells Serve as Phosphohydrolases at Physiological pH In Vivo: A Histochemical Implication

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.