Evidence for protein kinase C involvement in the regulation of renal 25-hydroxyvitamin D3-24-hydroxylase.

Although calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of various steroidogenic pathways, comparatively little is known of its role in the metabolism of vitamin D. The present study was undertaken to determine whether protein kinase C is involved in the regulation of renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, a major catabolic pathway for vitamin D metabolites in kidney and other target tissues. We examined the effect of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on 24-hydroxylase activity in fresh mouse renal tubules and correlated the changes in 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] production with translocation of protein kinase C and phosphorylation of mitochondrial proteins. PMA stimulated 24,25-(OH)2D3 synthesis, protein kinase C translocation from the cytosolic to the mitochondrial fraction, and phosphorylation of 30-35 K, 40 K, and 50 K mitochondrial proteins derived from 32P-labeled tubules. 4 alpha-Phorbol 12,13 didecanoate, an insert analog of PMA, did not elicit any of these effects. The synthetic diacylglycerol, oleoylacetyl glycerol, also stimulated 24,25-(OH)2D3 synthesis, whereas the protein kinase C inhibitors, H-7 and staurosporine, inhibited 24-hydroxylase activity. PMA did not further stimulate 24,25-(OH)2D3 production in tubules derived from mutant (Hyp) mice in which 24-hydroxylase and protein kinase C activities are elevated relative to normal. However, after treatment with H-7, 24-hydroxylase activity was reduced in both strains, and genotype differences were no longer apparent. Finally, H-7 failed to inhibit the induced renal 24-hydroxylase in tubules isolated from 1,25-dihydroxyvitamin D3-treated mice. These findings suggest a role for protein kinase C in the regulation of constitutive renal 24-hydroxylase and implicate the kinase in the aberrant expression of the hydroxylase in the Hyp mouse.