Phorbol ester synergizes with Ca²⁺ ionophore in activation of protein kinase C (PKC)α and PKCβ isoenzymes in human T cells and in induction of related cellular functions

Studies described herein were designed to examine the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA), and a Ca²⁺ ionophore (ionomycin), singly or in combination, on the activation and expression of the Ca²⁺-dependent protein kinase C (PKC) isoenzymes (α, β and γ) at the protein and messenger RNA (mRNA) levels in T cells. These two agents induce the activation and proliferation of T lymphocytes by mimicking the action of inositol phospholipid-derived second messengers normally generated by triggering of the antigen-specific T-cell receptor (TcR)/CD3 complex. TPA-induced T-cell proliferation, expression of interleukin-2 receptor-alpha subunit (IL-2Rα) and transferrin receptor, CD3 down-regulation and, lastly, the cytosol-to-membrane PKC translocation (determined by an enzymatic assay or by immunoblotting with a cross-reactive anti-PKC peptide antibody) were all facilitated by ionomycin. Immunoblots with isoenzyme-specific anti-PKC monoclonal antibodies demonstrated expression of immunoreactive PKCα, PKCβ and PKCγ proteins that were translocated to the membrane upon TPA plus ionomycin stimulation. Resting T cells expressed abundant levels of mRNA for PKCα and PKCβ, but very low levels (relative to brain) of PKCγ. TPA increased by two- to threefold the expression of PKCβ, but not of PKCα or PKCγ, mRNA within 12 hr of stimulation. Ionomycin synergized with TPA in increasing the expression of PKCα and PKCβ mRNA. The two agents also synergized in inducing expression of additional activation/growth-associated genes, namely the c-myc protooncogene, ornithine decarboxylase (ODC) and IL-2Rα. Ionomycin alone was inactive (or marginally active) in all of these assays. The translocation of distinct Ca²⁺-dependent PKC isoenzymes to the membrane and the up-regulation of PKCα and β mRNA suggest that at least these two isoenzymes are involved in discrete steps of the pathway leading to T-cell activation and proliferation. Moreover, the combined effects of TPA and ionomycin on T-cell function and cell-surface antigen expression appear to be due, at least in part, to their synergistic activation of distinct PKC isoenzyme(s).