Phorbol-12-myristate 13-acetate inhibits Nephronectin gene expression via Protein kinase C alpha and c-Jun/c-Fos transcription factors

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8β1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

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 (Fig. 3A). The effect of PMA on Npnt gene expression was also For quantification of ALP activity, cells were disrupted by sonication in 50 mM Tris-HCl containing 0.1% NP40. ALP activity was determined following incubation with the substrate p-nitrophenylphosphate and using absorbance at 405 nm. (b) For ALP staining, cells were fixed using 10% formalin in PBS and then ALP activity was visualized using a mixture of 0.1 mg/ml Naphthol As-Mx, 0.6 mg/ml phosphate, and Fast blue BB salt. (B) Total cellular RNA was extracted, then mRNA levels of Alp, Osteocalcin, and Gapdh were examined using quantitative real-time PCR analysis. Results are shown as the mean ± SD of three samples. **P < 0.01, Student's t test. www.nature.com/scientificreports/ examined and the results showed that expression to be significantly down-regulated by PMA (Fig. 3B). 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. 3C), while it was also significantly decreased by 10 nM of PMA at 12 h and then reached a plateau at 24 h (Fig. 3D). 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 downregulation 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. 4A), while down-regulation of Npnt gene expression by PMA was inhibited by Gö6983 (Fig. 4B). 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 downregulation 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. 4C), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 4D). 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 . Down-regulation of PKCα gene expression in MC3T3-E1 cells resulted in reduced phosphorylations of c-Jun and c-Fos (Suppl. Figure 2A,B). 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. 5A), down-regulation of Npnt gene expression by PMA was inhibited (Fig. 5B), and when c-Fos siRNA decreased the level of c-Fos (Fig. 5C), down-regulation of Npnt gene expression by PMA

Discussion
The present findings indicate that PMA, known to suppress osteoblast differentiation, downregulates Npnt gene expression. That downregulation was shown to be mediated via PKCα, and further via c-Jun and c-Fos, which are transcription factors in PKC signaling. Nakura    Reagents. PMA (phorbol 12-myristate 13-acetate) was purchased from Adipo Gen Life Sciences, Inc.

Statistical analysis.
Values are expressed as the mean ± SD. A two-sided unpaired Student's test was used for statistical analysis. Statistical differences were considered to be significant when the P value was < 0.05.