Participation of protein kinase C α in 1,25-dihydroxy-vitamin D 3 regulation of chick myoblast proliferation and differentiation

Abstract

Changes in morphology and DNA synthesis in cultured myoblasts in response to 1,25-dihydroxy-vitamin D 3 [1,25(OH) 2D 3] have previously suggested that the vitamin D hormone may affect muscle cell proliferation and differentiation. However, this interpretation was not substantiated by measurement of specific biochemical markers of myogenesis. To study the effect of 1,25(OH) 2D 3 on muscle development, chicken embryo myoblasts were cultured for 1–6 days in the presence or absence of 1,25(OH) 2D 3 (10 −9 M). The hormone increased DNA synthesis and decreased creatine kinase activity, indicating stimulation of cell proliferation and inhibition of myogenesis, in undifferentiated myoblasts (1 day of culture). At longer culture intervals, when myoblasts elongate and fuse to form differentiated myotubes, 1,25(OH) 2D 3 promoted myogenesis, as indicated by an inhibition of DNA synthesis and an increase in specific muscle differentiation markers as creatine kinase activity and myosin expression. The role of protein kinase C (PKC) in mediating the effects of hormone and the likely PKC isoform involved were also investigated. Increased PKC activity was observed during 1,25(OH) 2D 3 stimulation of myoblast proliferation whereas inhibition of PKC activity accompanied the effects of the hormone on myoblast differentiation. The specific PKC inhibitor calphostin suppressed hormone potentiation of DNA synthesis in proliferating myoblasts. 1,25(OH) 2D 3-dependent changes in the expression of PKC isoforms α, β, δ, ε and ζ during myogenesis were investigated by Western blot analysis. The early stimulation of myoblast proliferation by the hormone mainly correlated to increased PKC α expression whereas decreased PKC α levels were observed during the subsequent activation of myoblast differentiation. These results support that 1,25(OH) 2D 3 has a function in embryonic muscle growth and maturation, and PKC α may participate in the signal transduction pathway which mediates the response to the hormone.