Sclerostin Regulates Release of Bone Mineral by Osteocytes by Induction of Carbonic Anhydrase 2

Abstract

The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-kB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostinstimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats

“…Interestingly, a late expression of CA2 was seen at 18-M Sham rats contemporary with the increase in CTSK and SOST relative expressions. SOST was reported to induce catabolic activity via induction of CA2 and CTSK expression in osteocytes (Kogawa et al 2013). This finding suggests that osteocytic osteolysis plays a role in age-related bone loss rather than osteoclast-mediated resorption.…”

Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

“…OL enlargement was detected by lactation [17] in the presence of sclerostin [18] or microgravity [19], suggesting active regulation of the OL space and osteolysis. However, no direct evidence has yet been presented.…”

The role of carbonic anhydrase in the pathogenesis of vascular calcification in humans

“…These effects are reversed by acetazolamide. Consistent with this activity of sclerostin activating osteolysis, human trabecular bone samples treated with recombinant human sclerostin exhibit increased bone resorption, an effect that is reversed by acetazolamide [58].…”