We conclude that anti-apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p-Akt substrates and reduction of caspase activities. = 4 separate experiments. was abolished when osteoblasts were preincubated with pertussis toxin. We INCB024360 analog conclude that anti-apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p-Akt substrates and reduction of caspase activities. = 4 individual experiments. Unfavorable apoptosis and positive survival values indicate reduction and increase rates, respectively, from one representative experiment. * 0.001. ? 0.05. V-T, (control) vector transfected; siRNA-T, (VDR) siRNA transfected. Open in a separate window FIG. 1 1,25D treatment increases p-Akt levels in a dose- and time-dependent manner in ROS 17/2.8 osteoblasts. (A) Quantitative Western blot for the fold increase of p-Akt levels relative to total Akt obtained with different concentrations of the steroid hormone 1,25D for 10 min. Fold increase was normalized with respect to p-Akt/Akt values obtained in untreated cell cultures (0 nM 1,25D) made up of ethanol 0.01% as vehicle control. Vinculin was used as a control for protein loading (not shown). (B) Fold increase of p-Akt relative to total Akt levels and normalized to vehicle, obtained with 10 nM of hormone at the indicated time points. Data shown are mean values SE obtained in = 8 individual experiments.* 0.05, ** 0.001. 1,25D treatment reduces STSP-induced apoptosis through a PI3K/Akt signaling in ROS 17/2.8 osteoblasts To study the hypothesis that rapid upregulation of p-Akt by nanomolar concentrations of 1 1,25D leads to attenuation of apoptosis in osteoblasts, we measured the percentage of apoptotic ROS 17/2.8 cells with or without pretreatment with 10 nM 1,25D for 1 h. Apoptosis was induced with 100 nM STSP applied for 10 h after pretreating cells with either 10 nM 1,25D or vehicle (0.01% ethanol) for 1 h. As shown in Figs. 2A and 2B, 100 nM STSP induced a significant 2-fold increase in the percentage of apoptotic cells in osteoblast cultures at 80% confluence with respect to mock (untreated) cells. We found that pretreatment of ROS 17/2.8 cells with 10 nM 1,25D for 1 h before apoptosis induction decreased the number of apoptotic cells by 35% with respect to values obtained in the absence of hormone treatment (Table 1). To verify our hypothesis that a PI3K/Akt pathway is usually involved in 1,25D anti-apoptotic functions, we pretreated the cells with PI3K inhibitors wortmannin (50 nM) and LY294002 (10 M) for 30 min before incubation with 10 nM 1,25D. We found that pretreatment of ROS 17/2.8 cells with these inhibitors abolished 1,25D anti-apoptotic effects (Fig. 2B), thus confirming that 1,25D protection against apoptosis occurs through PI3K activation. Open in a separate window FIG. 2 1,25D treatment reduces staurosporine-induced apoptosis through activation of a PI3K pathway. (A and B) Flow cytometry data for the quantitative analysis of apoptotic ROS 17/2.8 cells with and without pretreatment with 10 nM 1,25D for 1 h, in the absence or presence of PI3K inhibitors wortmannin (50 nM) and LY294002 (10 M). Apoptotic cells were detected with PI staining of DNA. Apoptotic cells (sub-G1-gated events, M1) were defined as values corresponding to a DNA content 200 (2N) relative fluorescent units, as displayed around the horizontal axis of histograms. Apoptosis was induced with 100 nM STSP for 10 h. Mock cells were defined as cells grown with no apoptosis induction. Bar graph in B represents percent of apoptosis for the different treatments calculated from = 6 individual experiments. (C) ROS CCNF 17/2.8 cell density values of STSP-treated cultures, as measured with the CyQUANT NF cell proliferation assay (Invitrogen), with and without pretreatment with 10 nM 1,25D. = 5, * 0.05. RFU, relative fluorescence units. Cells were treated with 100 nM STSP 24 h after seeding (as indicated with the vertical arrow). Next, we measured cell densities of STSP-treated ROS 17/2.8 cultures for 3 days with or without pretreatment with 10 nM 1,25D. As shown in Fig. 2C, cell counts obtained from cultures pretreated with 1,25D were 30% higher than.Pertussis toxin-sensitive Gi protein and ERK-dependent pathways mediate ultrasound promotion of osteogenic transcription in human osteoblasts. with pertussis toxin. We conclude that anti-apoptotic effects of 1,25D in osteoblasts occur through nongenomic activation of a VDR/PI3K/Akt survival pathway that includes phosphorylation of multiple p-Akt substrates and reduction of caspase activities. = 4 individual experiments. Unfavorable apoptosis and positive survival values indicate reduction and increase rates, respectively, from one representative experiment. * 0.001. ? 0.05. V-T, (control) vector transfected; siRNA-T, (VDR) siRNA transfected. Open in a separate window FIG. 1 1,25D treatment increases p-Akt levels in a dose- and time-dependent manner in ROS 17/2.8 osteoblasts. (A) Quantitative Western blot for the fold increase of p-Akt levels relative to total Akt obtained with different concentrations of the steroid hormone 1,25D for 10 min. Fold increase was normalized with respect to p-Akt/Akt values obtained in untreated cell cultures (0 nM 1,25D) made up of ethanol 0.01% as vehicle control. Vinculin was used as a control for protein loading (not shown). (B) Fold increase of p-Akt relative to total Akt levels and normalized to vehicle, obtained with 10 nM of hormone at the indicated time points. Data shown are mean values SE obtained in = 8 individual experiments.* 0.05, ** 0.001. 1,25D treatment reduces STSP-induced apoptosis through a PI3K/Akt signaling in ROS 17/2.8 osteoblasts To study the hypothesis that rapid upregulation of p-Akt by nanomolar concentrations of 1 1,25D leads to attenuation of apoptosis in osteoblasts, we measured the percentage of apoptotic ROS 17/2.8 cells with or without pretreatment with INCB024360 analog 10 nM 1,25D for 1 h. Apoptosis was induced with 100 nM STSP applied for 10 h after pretreating cells with either 10 nM 1,25D or vehicle (0.01% ethanol) for 1 h. As shown in Figs. 2A and 2B, 100 nM STSP induced a significant 2-fold increase in the percentage of apoptotic cells in osteoblast cultures at 80% confluence with respect to mock (untreated) cells. We found that pretreatment of ROS 17/2.8 cells with 10 nM 1,25D for 1 h before apoptosis induction decreased the number of apoptotic cells by 35% with respect to values obtained in the absence of hormone treatment (Table 1). To verify our hypothesis that a PI3K/Akt pathway is usually involved in 1,25D anti-apoptotic functions, we pretreated the cells with PI3K inhibitors wortmannin (50 nM) and LY294002 (10 M) for 30 min before incubation with 10 nM 1,25D. We found that pretreatment of ROS 17/2.8 cells with these inhibitors abolished 1,25D anti-apoptotic effects (Fig. 2B), thus confirming that 1,25D protection against apoptosis occurs through PI3K activation. Open in a separate window FIG. 2 1,25D treatment reduces staurosporine-induced apoptosis through activation of a PI3K pathway. (A and B) Flow cytometry data for the quantitative analysis of apoptotic ROS 17/2.8 cells with and without pretreatment with 10 nM 1,25D for 1 h, in the absence or presence of PI3K inhibitors wortmannin (50 nM) and LY294002 (10 M). Apoptotic cells were detected with PI staining of DNA. Apoptotic cells (sub-G1-gated events, M1) were defined as values corresponding to a DNA content 200 (2N) relative fluorescent units, as displayed around the horizontal axis of histograms. Apoptosis was induced with 100 nM STSP for 10 h. Mock cells were defined as cells grown with no apoptosis induction. INCB024360 analog Bar graph in B represents percent of apoptosis for the different treatments calculated from = 6 individual experiments. (C) ROS 17/2.8 cell density values of STSP-treated cultures, as measured with the CyQUANT NF cell proliferation assay (Invitrogen),.