Protein kinase D Is an essential mediator during osteoblast differentiation

Abstract

Protein kinase D, also known as PKCp, is a serinelthreonine protein kinase that responds to phorbol esters and diacylglycerol. Using MC3T3-EI cells, we demonstrated that PKD is activated during DMSO-induced osteoblast differentiation. We further demonstrated that prolonged activation of protein kinase C pathways alone, i.e. in the absence of osteogenic inducers such as BMP-2 and/or DMSO, is sufficient to induce bone formation in preosteoblast cells in vitro. In this study, we examined the functional roles of PKD in osteoblastogenesis. Phorbol 12-myristate 13-acetate (PMA) activates most PKCs including PKD. Treatment of MC cells with PMA enhances osteoblast differentiation as indicated by increased ALP activity and bone nodules formation. Treatment with Gd6976, a specific inhibitor for PKD, at 0.02 and 0.2 pM resulted in a significant dose-dependent decrease in alkaline phosphatase (ALP) activity during osteoblast differentiation of MC cells mediated by DMSO, BMP2 or PMA. Bone nodule formation was attenuated simultaneously. Semi-quantitative RT-PCR analyses demonstrated that GO6976 treatment resulted in reduced Runx2, Osterix and ALP transcript expression in MC cells. However, a general PKC inhibitor GU6983 that does not inhibit PKD showed little effect on osteoblast differentiation, suggesting that PKD is crucial in the process. To confirm the functional roles of PKD, we overexpressed wild-type (PKD-WT), constitutively active (PKD-CA) and dominant negative (PKD-DN) PKD in MC cells and examine the effect of PKD modulation on osteoblast differentiation. In the absence of osteogenic inducers, MC cells transfected with PKD-CA resulted in enhanced bone nodules formation as compared to the PKD-DN transfectants. These findings are in good concordance to our previous observations that PKD activation alone is sufficient for inducing osteoblast differentiation. Using both pharmacological and PKD overexpression studies, our findings suggested that PKD plays essential roles in osteoblast differentiation and that osteoblast differentiation can be modulated via the activation of PKD.