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As shown in Fig. 5D, Car2 siRNA transfection prevented the removal of calcein and decrease in fluorescence intensity in response to rhSCL. Together, these data demonstrate that Car2 expression in MLO-Y4 is induced by rhSCL and that CA2 plays a direct role in the resulting loss of calcium from mineralized substrates, strongly implying that sclerostin-induced http://www.selleckchem.com/products/z-vad-fmk.html CA2 plays a role in osteocytic osteolysis. To investigate which of the reported receptors for sclerostin were important for the response to sclerostin, we used siRNA to knock down individually the expression of Lrp4, Lrp5, and Lrp6 mRNA. The extent of knockdown in their expression was 76%, 52%, and 65%, respectively, after 24 hours and was confirmed to be specific for each of the Lrps (Supplemental Fig. S1). Knockdown of each of these receptors inhibited the induction of Car2 mRNA expression by sclerostin (Fig. 6A). Examination of other genes potentially related to an osteocytic osteolysis effect revealed that induction by sclerostin of both Catk and Acp5 mRNA was also abrogated by knockdown of all receptors (Fig. 6B, C). Interestingly, the induction by sclerostin of Clcn7 mRNA expression was unaffected by Lrp4 knockdown but was abrogated in the case of Lrp5 and Lrp6 knockdown (Fig. 6D). Consistent with these effects, knockdown of each receptor resulted in the abolition of an effect of sclerostin http://www.selleck.cn/products/BIBW2992.html on both pHo change (Fig. 6E) and calcium release (Fig. 6F), both implicating these receptors in the sclerostin response in a nonredundant fashion and the interruption of the ��-catenin signaling pathway in this response. To test whether sclerostin induction of CA2 resulted in an effect on osteocyte activity in situ, human trabecular bone obtained from patients undergoing hip replacement surgery was cultured ex vivo for up to 7 days in the presence or absence of rhSCL. Resulting histological assessment of decalcified sections of rhSCL-treated bone revealed a significant increase http://www.selleckchem.com/products/ly2157299.html in the mean osteocyte lacunar area (Lac.Ar) compared with bone incubated in control medium (Fig. 7). The increase in lacunar size was reversed in the co-presence of acetozolamide (Fig. 7), suggesting that the sclerostin-induced increase in CA2 levels may be a component of osteocytic osteolysis in human bone. Analysis of SOST-Tg mice bone revealed a significant increase in mean Lac.Ar in cortical bone compared with that in age-matched WT littermates (WT?=?7.5?��?0.1?��m2 versus Sost-Tg?=?8.0?��?0.1?��m2, p?