Cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC):: Response to genistein and pyrrolidine dithiocarbamate

Using an immortalized astrocyte cell line (DITNC), we showed that lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) but not interferon-alpha (IFN-alpha) could individually induce secretory phospholipase A(2) (sPLA(2)) mRNA and enzymatic activity. However, induction of inducible nitric oxide synthase (iNOS) mRNA and NO production by cytokines required the presence of IFN-gamma. Using a three-cytokine mixture (TNF-alpha, IL-1 beta, and IFN-gamma) that could maximally induce both iNOS and sPLA(2), the increase in these mRNA species reached a maximum by 4-8 h, followed by a decline up to 48 h. L-N-6-(1-Iminoethyl)lysine acetate (L-NIL) inhibited cytokine-induced NO production with IC50 of 25 mu M, but this compound did not affect iNOS mRNA, Furthermore, L-NIL, exerted no effect on sPLA(2) mRNA or sPLA(2) activity. Pyrrolidine dithiocarbamate (PDTC), an inhibitor for NF-kappa B, was more effective in inhibiting iNOS mRNA and NO production than for sPLA(2). Surprisingly, genistein inhibited both NO production and sPLA(2) activity with IC50 of 72 mu M and 88 mu M, respectively. On the other hand, daidzein, a genistein analog lacking tyrosine kinase inhibitor activity, was not effective in inhibition of NO production at 250 mu M. These results demonstrate distinct pathways for induction of iNOS and sPLA(2) in DITNC cells by cytokines and shed new insight on transcriptional regulation for these two mRNA species.