1,25-Dihydroxyvitamin D₃ and TPA activate phospholipase D in Caco-2 cells: Role of PKC-α

1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 12-O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF- 109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)₂D₃ activated PKC-α, but not PKC-β₁, -β(II), -δ, or -ζ, whereas TPA activated PKC-α, -β₁, and - δ. Chronic treatment with TPA (1 μM, 24 h) significantly reduced the expression of PKC-α, -β(I), and -δ and markedly reduced the ability of 1,25(OH)₂D₃ or TPA to acutely stimulate PLD. Removal of Ca²⁺ from the medium, as well as preincubation of cells with Go-6976, an inhibitor of Ca²⁺-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)₂D₃ or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20- acetate, which specifically activates PKC-β(I) and -β(II), however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)₂D₃ or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-α expression, respectively. Taken together, these observations indicate that PKC-α is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)₂D₃ or TPA.