1,25(OH) 2-Vitamin D 3 Affects the Subcellular Distribution of Protein Kinase C Isoenzymes in Muscle Cells

Abstract

Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D 3 [1,25(OH) 2D 3] regulation of DNA synthesis (long-term effect) and Ca 2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cell proliferation and growth by 1,25(OH) 2D 3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the presence of PKC isoenzymes in chicken embryo myoblasts and changes in their total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH) 2D 3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 μM) were investigated. Western blot analysis provided evidence on the expression of PKC α, β and δ isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) were detected for each isoenzyme. 1,25(OH) 2D 3 and DOG, which increased myoblast PKC activity parallel with the stimulation of DNA synthesis and culture growth and the phorbol ester TPA which induced the opposite changes, exerted differential effects on PKC isoenzymes. Long-term (72 h) treatment with 1,25(OH) 2D 3 and DOG did not change total PKC isoform levels but decreased the 80 kDa species and increased the release of the catalytic fragment of PKC δ and β, whereas TPA augmented the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC β and δ and the 50 kDa species for PKC α. Subcellular distribution studies showed that the 80 kDa molecule is only present in the cytosolic fraction whereas in the particulate fractions the 50 kDa fragments are detected. Increased amounts of the catalytic fragments of PKC β and δ both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH) 2D 3 increases PKC β in the nucleus and PKC δ in membranes. TPA induced the appearance of the 50 kDa species of PKC α in the nuclear and membrane fractions. The phorbol ester also decreased the catalytic fragments of PKC β and δ in membranes. Increased levels of PKC β, and to a lesser extent of PKC δ, in membranes and cytosol could be detected after short exposure (5 min) of myoblasts to 1,25(OH) 2D 3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca 2+-dependent PKCs α and β and the Ca 2+-independent PKC δ. Moreover, the results suggest that the β and δ isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH) 2D 3.