PMA suppresses BMP-2 induced osteoblast differentiation in MC3T3-E1 cells.
To investigate the effect of PMA on osteoblastic differentiation, MC3T3-E1 cells were cultured with BMP-2 (100 ng/ml) in the absence or presence of PMA (5 nM) for three days. ALP activity in cells cultured with BMP-2 was shown to be increased, whereas it was significantly suppressed when cells were cultured in the combination of BMP-2 and PMA (Fig. 1Aa, b). At the same time, the gene expressions of Alp and Osteocalcin, differentiation markers of osteoblasts, were investigated. Both Alp and Osteocalcin gene expressions induced by BMP-2 were suppressed by PMA. These results showed that PMA suppressed BMP-2 induced osteoblast differentiation (Fig. 1B) 26.
Npnt gene expression is suppressed by PMA in dose and time-dependent manner.
PMA, a phorbol ester, is known to activate the PKC signaling pathway. To determine whether PMA activated the PKC signaling pathway in MC3T3-E1 cells, Marcks phosphorylation was examined, as previous studies have reported that it was phosphorylated by PKC activation 27,28. Marcks was remarkably phosphorylated by PMA (Fig. 2A). The effect of PMA on Npnt gene expression was also examined and the results showed that expression to be significantly down-regulated by PMA (Fig. 2B). Next, the effects of PMA on dose- and time-dependent Npnt gene expression were investigated. That expression was significantly decreased by PMA at 3.2 nM and reached a plateau at 32 nM (Fig. 2C), while it was also significantly decreased by 10 nM of PMA at 12 hours and then reached a plateau at 24 hours (Fig. 2D). These results suggest that Npnt gene expression is suppressed by PMA in a dose and time-dependent manner.
PKCα is involved in down-regulation of Npnt gene expression by PMA.
To verify whether down-regulation of Npnt gene expression by PMA is involved in the PKC signaling pathway, MC3T3-E1 cells were pretreated with Gö6983, known as a broad-spectrum PKC inhibitor, before PMA stimulation. Phosphorylation of Marks by PMA did not occur following pretreatment with Gö6983 (Fig. 3A), while down-regulation of Npnt gene expression by PMA was inhibited by Gö6983 (Fig. 3B). These results suggest that Npnt gene expression is involved in the PKC signaling pathway.
It has been reported that PKCα is highly expressed in MC3T3-E1 cells 29. To verify its involvement in down-regulation of Npnt gene expression, MC3T3-E1 cells were pretreated with or without Pkcα siRNA, and thereafter with PMA alone or in combination. When Pkcα siRNA decreased the cellular protein level of Pkcα (Fig. 3C), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 3D). These results indicate that PKCα is involved in down-regulation of Npnt gene expression by PMA.
Both of c-Jun and c-Fos are involved in down-regulation of Npnt gene expression.
It has been reported that regulation of gene expression by PMA is involved in activation of PKCα and thereafter of AP-1 30. To investigate the involvement of c-Jun and c-Fos as transcription factors, which compose AP-1, on down-regulation of Npnt gene expression, MC3T3-E1 cells were pretreated with or without c-Jun, c-Fos siRNA, and then treated with PMA alone or in combination. When c-Jun siRNA decreased the cellular protein level of c-Jun (Fig. 4A), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 4B), and when c-Fos siRNA decreased the level of c-Fos mRNA (Fig. 4C), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 4D). These results demonstrated that the transcription factors c-Jun and c-Fos are involved in down-regulation of Npnt gene expression by PMA.