The expression profile of PKC isoforms during MC3T3-E1 differentiation

Protein kinase C (PKC) is a family of kinases whose isoforms show subtle differences in physiological and biochemical responses, with their expression being cell- specific. We hypothesize that there may be a specific profile of expression of PKC isoforms in differentiating osteoblastic cells (OBC) with individual isoforms having specific functions. Herein, the MC3T3-E1 cell line was used as a differentiating model, which was induced from the pre-osteoblast stage to mature osteoblast and characterized with several phenotypic markers, including alkaline phosphatase activity, osteocalcin and bone sialoprotein. The expression of PKC isoforms was monitored using Western blot analysis. Upon induction of osteogenesis, the intracellular localization of PKC eta and theta was determined using immunofluorescence. Lastly, the effect of P38 MAP kinase inhibition was determined using SB203580. Results show 1) PKC alpha, delta, lambda were all highly expressed in MC3T3-E1 osteoblastic cells, 2) the expression of PKC theta was significantly down-regulated upon induction of osteoblastic differentiation; 3) PKC eta was non-detectable at certain cell culture days; however, was up-regulated as the cells transit from each differentiation phase. The increased expression of PKC eta correlated with increases in OC, BSP levels and alkaline phosphatase activity. Immunofluorescence procedure confirmed the Western blot results with an increase in PKC eta and a decrease in PKC theta upon osteogenic stimulation. The inhibition of p38 resulted in a marked down-regulation of PKC eta. The data demonstrate that there is a specific profile of expression of PKC isoforms in differentiating osteoblasts; the different expression pattern of individual isoforms may be either a consequence of the differentiation itself or plays a role in the regulatory mechanism of osteoblastic differentiation. This study has provided primary information on the temporal pattern of expression of PKC isoforms in the differentiating osteoblast and further insight into their possible role in osteoblastic cell maturation.