STIMULATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN RAT-LIVER BY HYALURONAN FRAGMENTS

Hepatic injury and chronic wounding are characterized by increased syn thesis of extracellular matrix proteins including hyaluronan (HA). Rec ently, it has been recognized that low-molecular-weight fragments of H A, but not native HA (e.g., high-molecular-weight HA), induce inflamma tory gene expression, and activate the transcriptional regulator, nucl ear factor kappa B (NF-kappa B). The inducible isoform of nitric oxide synthase (iNOS) is induced by cytokines and/or lipopolysaccharide (LP S) through the NF-kappa B signal transduction pathway. Because of this association, we hypothesized that HA fragments might also stimulate i NOS gene transcription. The aims of this study were therefore to deter mine whether HA or HA fragments induced iNOS in hepatic cells, and to characterize the signaling pathway. HA fragments (100 mu g/mL) markedl y stimulated iNOS messenger RNA (mRNA) in endothelial and Kupffer cell s, but minimally induced this mRNA in hepatocytes and stellate cells. High-molecular-weight HA (200 mu g/mL) had no effect on iNOS mRNA in a ny cell type. The addition of interferon gamma (IFN-gamma) to HA fragm ents resulted in stimulation of iNOS mRNA 2-, 3-, 4-, and 10-fold abov e that for HA fragments alone in hepatocytes, endothelial, Kupffer, an d stellate cells, respectively. The combination of HA. fragments and L PS did not result in an incremental increase in iNOS mRNA induction. i NOS protein and nitrite levels (used as a measure of NO production and NOS enzymatic activity) paralleled closely iNOS mRNA expression and i ncreased proportionally to HA. fragment concentration in a dose-depend ent fashion. At 1 hour following stimulation, NF-kappa B DNA binding a ctivity was detected in extracts from Kupffer cells stimulated with HA fragments, but not in those exposed to media alone or to high-molecul ar-weight HA. Finally, inhibitors of NF-kappa B blocked HA fragment-de pendent iNOS mRNA induction in Kupffer and sinusoidal endothelial cell s. The data indicate that HA fragments, but not high-molecular-weight HA, induce iNOS in liver, having the greatest effects on endothelial a nd Kupffer cells. We speculate that HA fragments may be an important s timulus for NO production in various forms of liver disease, particula rly as a cofactor with inflammatory cytokines.