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

Abstract

Using an immortalized astrocyte cell line (DITNC), we showed that lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) but not interferon-alpha (IFN-alpha) could individually induce secretory phospholipase A2 (sPLA2) 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-1beta, and IFN-gamma) that could maximally induce both iNOS and sPLA2, the increase in these mRNA species reached a maximum by 4-8 h, followed by a decline up to 48 h. L-N6-(1-Iminoethyl)lysine acetate (L-NIL) inhibited cytokine-induced NO production with IC50 of 25 microM, but this compound did not affect iNOS mRNA. Furthermore, L-NIL exerted no effect on sPLA2 mRNA or sPLA2 activity. Pyrrolidine dithiocarbamate (PDTC), an inhibitor for NF-kappaB, was more effective in inhibiting iNOS mRNA and NO production than for sPLA2. Surprisingly, genistein inhibited both NO production and sPLA2 activity with IC50 of 72 microM and 88 microM, respectively. On the other hand, daidzein, a genistein analog lacking tyrosine kinase inhibitor activity, was not effective in inhibition of NO production at 250 microM. These results demonstrate distinct pathways for induction of iNOS and sPLA2 in DITNC cells by cytokines and shed new insight on transcriptional regulation for these two mRNA species.