1,25(OH)(2)-VITAMIN-D-3 AFFECTS THE SUBCELLULAR-DISTRIBUTION OF PROTEIN-KINASE-C ISOENZYMES IN MUSCLE-CELLS

Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D-3 [1,25(OH)(2)D-3] regulation of DNA synth esis (long-term effect) and Ca2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cel l proliferation and growth by 1,25(OH)(2)D-3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the pre sence of PKC isoenzymes in chicken embryo myoblasts and changes in the ir total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH)(2)D-3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA ; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 mu M) were investig ated. Western blot analysis provided evidence on the expression of PKC alpha, beta and delta isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) wer e detected for each isoenzyme. 1,25(OH)(2)D-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 opposit e changes, exerted differential effects on PKC isoenzymes. Longterm (7 2 h) treatment with 1,25(OH)(2)D-3 and DOG did not change total PKC is oform levels but decreased the 80 kDa species and increased the releas e of the catalytic fragment of PKC delta and beta, whereas TPA augment ed the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC beta and delta and the 50 kDa species for PKC or. Subce llular distribution studies showed that the 80 kDa molecule is only pr esent in the cytosolic fraction whereas in the particulate fractions t he 50 kDa fragments are detected. Increased amounts of the catalytic f ragments of PKC beta and delta both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH)(2)D-3 increases PKC beta in the nucleus and PKC delta in membranes. TPA induced the ap pearance of the 50 kDa species of PKC alpha in the nuclear and membran e fractions. The phorbol ester also decreased the catalytic fragments of PKC beta and delta in membranes. Increased levels of PKC beta, and to a lesser extent of PKC delta, in membranes and cytosol could be det ected after short exposure (5 min) of myoblasts to 1,25(OH)(2)D-3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca2+-dependent PKC s alpha and beta and the Ca2+-independent PKC delta. Moreover, the res ults suggest that the beta and delta isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH)(2)D-3. (C) 19 98 Elsevier Science Inc.