Hepatic injury and chronic wounding are characterized by increased synthesis of extracellular matrix proteins including hyaluronan (HA). Recently, it has been recognized that low-molecular-weight fragments of HA, but not native HA (e.g., high-molecular-weight HA), induce inflammatory gene expression, and activate the transcriptional regulator, nuclear factor kappaB (NF-kappaB). The inducible isoform of nitric oxide synthase (iNOS) is induced by cytokines and/or lipopolysaccharide (LPS) through the NF-kappaB signal transduction pathway. Because of this association, we hypothesized that HA fragments might also stimulate iNOS gene transcription. The aims of this study were therefore to determine whether HA or HA fragments induced iNOS in hepatic cells, and to characterize the signaling pathway. HA fragments (100 microg/mL) markedly stimulated iNOS messenger RNA (mRNA) in endothelial and Kupffer cells, but minimally induced this mRNA in hepatocytes and stellate cells. High-molecular-weight HA (200 microg/mL) had no effect on iNOS mRNA in any cell type. The addition of interferon gamma (IFN-gamma) to HA fragments resulted in stimulation of iNOS mRNA 2-, 3-, 4-, and 10-fold above that for HA fragments alone in hepatocytes, endothelial, Kupffer, and stellate cells, respectively. The combination of HA fragments and LPS did not result in an incremental increase in iNOS mRNA induction. iNOS protein and nitrite levels (used as a measure of NO production and NOS enzymatic activity) paralleled closely iNOS mRNA expression and increased proportionally to HA fragment concentration in a dose-dependent fashion. At 1 hour following stimulation, NF-kappaB DNA binding activity was detected in extracts from Kupffer cells stimulated with HA fragments, but not in those exposed to media alone or to high-molecular-weight HA. Finally, inhibitors of NF-kappaB blocked HA fragment-dependent iNOS mRNA induction in Kupffer and sinusoidal endothelial cells. The data indicate that HA fragments, but not high-molecular-weight HA, induce iNOS in liver, having the greatest effects on endothelial and Kupffer cells. We speculate that HA fragments may be an important stimulus for NO production in various forms of liver disease, particularly as a cofactor with inflammatory cytokines.
