Induction of iNOS expression in skeletal muscle by IL-1β and NFκB activation: an in vitro and in vivo study

OBJECTIVE: The intracellular pathway and the regulation of inducible nitric oxide synthase (iNOS) expression in skeletal muscle is incompletely understood. In vitro studies, using different cell types, suggest that inflammatory cytokines are potential triggers to induce iNOS expression. METHODS: To analyze intracellular pathways leading to iNOS induction, rat skeletal myoblasts were incubated with inflammatory cytokines and assessed for iNOS expression by Western blot and Griess reaction. To confirm the in vitro findings, local cytokine levels were determined in skeletal muscle biopsies of patients with chronic heart failure (CHF) and correlated with iNOS expression. RESULTS: Nitrite accumulation in the myoblast culture supernatant or iNOS protein in the cell pellet was significantly increased after incubation with IL-1beta in combination with gamma-IFN. Priming experiments revealed that gamma-IFN elevated the expression of IL-1beta receptor mRNA, whereby IL-1beta was able to induce iNOS expression. The cytokine-mediated iNOS induction was significantly reduced by blocking ERK1/ERK2 activation and completely abolished by the inhibition of NFkappaB. In skeletal muscle biopsies of CHF patients the local content of IL-1beta was significantly increased as compared to healthy controls. Furthermore, a linear correlation between IL-1beta content and iNOS expression in the skeletal muscle was detected. CONCLUSIONS: These data demonstrate that IL-1beta, together with the priming effect of gamma-IFN, induces iNOS expression in skeletal muscle via activation of ERK1/ERK2 and NFkappaB.