Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism

Abstract

The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.

Sclerostin Stimulates Osteocyte Support of Osteoclast Activity by a RANKL-Dependent Pathway

“…It is also possible that transgenic chronic overexpression of Sost triggers a negative feedback effect, which counters the effect on osteoclast activity. This is supported by our observations in human primary osteocyte-like cells that SCL only transiently upregulated the RANKL:OPG mRNA ratio cells, and by our published observation that SCL down-regulates the expression at least one of the known receptors for SCL, LRP4 [36]. It will be of interest in future studies to examine acute versus chronic administration of sclerostin in vivo.…”

“…We previously showed that human osteocytelike cells were sensitive to the anti-anabolic effects of SCL at concentrations as low as 1 ng/ml, concentrations that are higher than but near to the levels reported in human serum of between 0.3 and 0.6 ng/ml [49,50,51]. This suggested that cells at the preosteocyte and osteocyte stages are major physiological targets for SCL [36]. In the present study, we found that SCL levels of 10 ng/ml or higher produced catabolic activity in these cells, increasing RANKL and decreasing OPG expression.…”

“…Cells were cultured in triplicate in wells of a 96 well plate at 8610 3 cells/well for NHBC or 5610 3 cells/well for cell lines, in aMEM-10 containing dexamethasone (10 28 M) and KH 2 PO 4 (1.8 mM) in the presence or absence of rhSCL. Mature, late osteoblast/osteocyte-like cultures of NHBC were generated by culturing for 35 days in mineralisation medium, as previously described [36]. Cells were then treated with rhSCL (1, 10 or 50 ng/ml) for 3 or 7 days, and RNA prepared as described above.…”

“…Because late-stage cultures exhibited greater sensitivity to sclerostin in terms of osteoclastogenesis-associated gene expression and because we have identified late osteoblasts/preosteocytes as SCL targets [36], we next cultured cells under differentiating conditions for 35 days and then treated these acutely with rhSCL. rhSCL dose-dependently increased RANKL mRNA expression after 3 days (mean fold-change with rhSCL at 50 ng/ml of 9.764.7, n = 3) with levels returning thereafter to those of control cultures in all cases, while the lowest dose of rhSCL used (1 ng/ml) had no effect ( Fig.…”

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

“…Sclerostin was also significantly decreased in the serum of HMWKO mice. Sclerostin also functions to inhibit bone formation through modulation of the Phex/Mepe ASARM pathway (38) by increasing Mepe-ASARM protein that competitively binds with Phex inhibiting activation of Dmp1 (26). Activated Dmp1 is able to nucleate hydroxyapatite crystals during the initiation of bone formation (39).…”