FIGURE 5.
TNFRSF19 activates osteogenesis by increasing ALP activity. A, TNFRSF19 is up-regulated upon Wnt3a treatment. hMSC-LRP5T253 (T253) cells were treated with 50% control condition medium (Co-CM) or Wnt3a conditioned medium (Wnt3a-CM) for the indicated hours. The expression of osteogenic marker ALP and TNFRSF19 transcripts 1 and 2 (TNFRSF19.1 and TNFRSF19.2) were analyzed by real-time RT-PCR and normalized against GAPDH. Results are mean ± S.D. of three replicates. B, overexpression of TNFRSF19.2 restores ALP activity in hMSC-LRP5T244 (T244) cells. T244 and T253 cells were infected with pBABE empty vector or TNFRSF19.2 expression vector followed by culturing in normal medium, control CM, or Wnt3a-CM for 7 days. ALP activity was measured by using p-nitrophenyl phosphate as substrate and normalized against cell number. Results are mean ± S.D. of five replicates. *, p < 0.05; **, p < 0.01. C, knocking down TNFRSF19 in T253 cells decreased Wnt3a-induced ALP activity. T253 cells were infected with control shRNA (shCtrl) or three shRNAs targeting TNFRSF19 (sh19-2, sh19-4, or sh19-5). Results are mean ± S.D. of five replicates. *, p < 0.05; **, p < 0.01 compared with shCtrl.