Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1.beta. gene expression

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Abstract
Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., and Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1.beta. (IL-1.beta.) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 .times. 10-8 M TPA. Higher concentratios of TPA, which partially or totally depleted protein kinase C levels in the cells (10-9-2 .times. 10-5 M), had an inhibitory effect on IL-1.beta. mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1.beta. mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 .times. 10-8 M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the .alpha., .beta., and .gamma. isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC .alpha. was particulate, and PKC .beta. was cytosolic, while there was no detectable PKC .gamma.. Upon TPA treatment, there was a time-dependent translocation (maximum 1 h) of PKC .alpha. to a particulate compartment, followed by its gradual disappearance (70% by 3 h, not detectable by 6 h), with no concomitant rise in the cytosolic form. PKC .beta. remained cytosolic during TPA treatment, while PKC .gamma. appeared at 6 h and continued to increase in abundance by 24 h, mostly in particulate form. Exposure of 32PO4-labeled cells to TPA (5 .times. 10-8 M) for 30 min enhanced the phosphorylation of several major substrates; 5 of 10 were in the same postmitochondrial fraction into which PKC .alpha. translocated. Exposure of U937 cells to diC8 (5 .times. 10-5-10-4 M) failed to induce PKC .alpha. translocation. Although diC8 induced the phosphorylation of five substrates, these were cytosolic and were distinct from the substrates phosphorylated in the presence of TPA. D-Sphingosine of H-7, PKC antagonists, prevented the accumulation of IL-1.beta. transcripts and TPA-induced phosphorylation of endogenous substrates in a dose-dependent manner. Our data suggest a potential role for PKC .alpha. mediated phosphorylation of substrates in the initial events leading to TPA-induced differentiation of a promonocytic cell to a monocyte/macrophage.