Inhibition of DNA synthesis in cultured hepatocytes by endotoxin-conditioned medium of activated stellate cells is transforming growth factor-beta and nitric oxide-independent.

Abstract

Activated hepatic stellate cells play a major role in the pathophysiology of chronic liver disease. They can influence the metabolism of hepatocytes by producing a variety of cytokines and growth factors. Upon stimulation with endotoxin, stellate cells also synthesize nitric oxide (NO), a potent mediator of growth of several cell types including hepatocytes. We investigated the effect of serum-free medium conditioned by activated stellate cells in the absence and presence of endotoxin on NO and DNA synthesis in hepatocytes. Stellate cells and hepatocytes were isolated by enzymatic digestion of the liver. Stellate cells were cultured for 10 days after which the majority exhibited alpha-smooth muscle actin (a marker for activated cells); hepatocytes were used after overnight culture. While the medium conditioned by stellate cells in the absence of endotoxin stimulated DNA synthesis in hepatocytes, medium conditioned in its presence inhibited this process in an endotoxin concentration-dependent manner (10 - 1000 ng ml(-1)). Endotoxin-conditioned stellate cell medium also stimulated NO synthesis in hepatocytes; the effect was consistent with increased protein and mRNA expression of inducible NO synthase (iNOS). However, inhibition of DNA synthesis in hepatocytes caused by endotoxin-conditioned stellate cell medium was unaffected by the NOS inhibitor, L-N(G)-monomethylarginine (L-NMMA), guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and neutralizing antibodies for TGF-beta, IL-1beta, IL-6 and TNF-alpha. These results indicate that factors other than these cytokines produced by activated stellate cells upon stimulation with endotoxin or by hepatocytes challenged with endotoxin-conditioned stellate cell medium inhibit DNA synthesis in hepatocytes.