PROPERTIES OF PROTEIN-KINASE-C ISOFORMS (BETA-II, EPSILON, AND ZETA) IN A MACROPHAGE CELL-LINE (J774) AND THEIR ROLES IN LPS-INDUCED NITRIC-OXIDE PRODUCTION

Northern analysis of poly(A)(+) RNA extracted from J774 cells (a mouse macrophage cell line) showed that this cell line constitutively expre sses mRNAs specific for protein kinase C (PKC)-beta I, -beta II, -epsi lon and -zeta, but not those for PKC-alpha, -gamma or -delta. Western analysis of the total cell lysate showed that J774 cells express PKC-b eta II, -epsilon and -zeta isoenzymes, but failed to show the expressi on of PKC-beta I. The exposure of J774 cells to >10 nM PMA led to a lo ss of immunoreactive PKC-beta II in 4 h. The down-regulation of immuno reactive PKC-epsilon required more than 8 h of the exposure to >100 nM PMA. Immunoreactive PKC-zeta was most resistant to PMA treatment and was not significantly reduced after the exposure to 300 to 600 nM PMA for 24 h. PMA-mediated, persistent down-regulation of PKC-PII is proba bly a result of the inhibition of PKC-PII biosynthesis at the posttran scriptional level, because PMA-exposed cells were found to gradually i ncrease the expression of PKC-PII specific mRNA. PMA-pretreated cells responded to a low dose (10 ng/ml), but not to a high dose (1 mu g/ml) , of LPS by significantly lower expression of mRNA specific for the in ducible nitric oxide synthase (i-NOS) gene and production of nitric ox ide (NO) than the control cells did. Thus, PKC could be a part of the signal transduction apparatus involved in LPS-induced inducible nitric oxide synthase gene activation.