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 expresses mRNAs specific for protein kinase C (PKC)-beta I, -beta II, -epsilon and -zeta, but not those for PKC-alpha, -gamma or -delta. Western analysis of the total cell lysate showed that J774 cells express PKC-beta II, -epsilon and -zeta isoenzymes, but failed to show the expression of PKC-beta I. The exposure of J774 cells to > 10 nM PMA led to a loss of immunoreactive PKC-beta II in 4 h. The down-regulation of immunoreactive 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-beta II is probably a result of the inhibition of PKC-beta II biosynthesis at the posttranscriptional level, because PMA-exposed cells were found to gradually increase the expression of PKC-beta II specific mRNA. PMA-pretreated cells responded to a low dose (10 ng/ml), but not to a high dose (1 microgram/ml), of LPS by significantly lower expression of mRNA specific for the inducible nitric oxide synthase (i-NOS) gene and production of nitric oxide (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.