Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation

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Abstract
In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-α, -γ, -δ, -ε, -ζ and -µ. However we were unable to detect PKC-β, -η or -θ. The same PKC isoform expression profile was found in rat adipocytes. The α, δ and γ isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-ε increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-µ increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-ζ levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-ζ but that PKC-α,-δ and -µ each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-ε and -γ revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-γ, but not PKC-ε, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-ε is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of the adipocyte phenotype.