Fig. 3. CSNK2B deficiency in osteocytes upregulates sclerostin expression.

a, b WT BMSCs were cultured under osteogenic conditions in the presence of the conditioned medium (CM) harvested from shScr- or shCsnk2b-expressing Ocy454 cells, and six days later, ALP activity (a) and osteogenic gene expression (b) were analyzed. c–e The CM harvested from shScr- or shCsnk2b-expressing Ocy454 cells six days after differentiation was added to mouse BMMs and cultured under osteoclast differentiation condition for 12 days. Osteoclast differentiation was assessed by TRAP staining (c), TRAP activity (d), and osteoclast gene expression (e). Scale bar, 50 μm (c). f Heat map showing differential gene expression in shScr- and shCsnk2b-expressing Ocy454 cells. Top 30 genes with upregulated or downregulated expression are presented (triplicates/group). The log10 expression (read count) was centered across samples. Red and purple denote upregulation and downregulation, respectively. (absolute log2 fold change >1.2 in Csnk2b-deficient osteocytes as compared to control). Arrows indicate upregulated expression of Sost and Phex and knockdown of Csnk2b expression in Csnk2b-deficient osteocytes as compared to control. g Immunoblot analysis showing protein levels of sclerostin and CSNK2B in shScr- and shCsnk2b-expressing Ocy454 cells six days after osteocyte differentiation (top) and in Csnk2b^fl/fl and Csnk2b^Dmp1 tibias (bottom). h Immunohistochemistry for sclerostin in the cortical bone of eight-week-old Csnk2b^fl/fl and Csnk2b^Dmp1 femurs (top). Numbers of sclerostin-expressing osteocytes per total osteocytes were quantitated (bottom). IgG was used for negative staining. Scale bar, 20 μm (top). M muscle, BM bone marrow, CB cortical bone. Arrows indicate sclerostin-expressing osteocytes. i Axin2 mRNA levels in 8-week-old Csnk2b^fl/fl and Csnk2b^Dmp1 tibias were assessed by RT-PCR and normalized to Rplp0 (n = 4/group). j C3H10T1/2 cells were transfected with the TopFlash reporter gene along with Renilla and incubated with the CM obtained from shScr- or shCsnk2b-expressing Ocy454 cells. After 48 h, luciferase activity was measured and normalized to Renilla. k WT BMSCs were treated with CM of shScr- or shCsnk2b-expressing Ocy454 cells under osteogenic differentiation condition, Axin2 mRNA levels were assessed at day 6 by RT-PCR (n = 4/group). Data are representative of three (a–e, g–k). A two-tailed unpaired Student’s t-test for comparing two groups (a, b, d, e, h–k; error bars, SD of biological replicates).