Obligatory role of heat shock protein 90 in iNOS induction

Luo S, Wang T, Qin H, Lei H, Xia Y. Obligatory role of heat shock protein 90 in iNOS induction. Am J Physiol Cell Physiol 301: C227-C233, 2011. First published March 23, 2011; doi:10.1152/ajpcell.00493.2010.-Inducible nitric oxide (NO) synthase (iNOS) plays an important role in cell injury and host defense. While undetectable in normal tissues, iNOS expression is induced by endotoxins and inflammatory cytokines primarily via the I kappa B kinase/nuclear factor-kappa B (IKK-NF-kappa B) and Janus kinase (JAK)-signal transducers and activators of transcription 1 (STAT1) pathways. Our previous studies found that heat shock protein 90 (Hsp90) associates with iNOS, and this association enhances iNOS activity. Here we show that Hsp90 is also essential for iNOS induction. With mouse macrophages, Hsp90 inhibition by geldanamycin or knockdown with small interfering RNA (siRNA) prevented lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma)-stimulated iNOS protein expression. RT-PCR experiments showed that iNOS mRNA transcription was blocked by Hsp90 inhibition. Radicicol, another Hsp90 inhibitor whose structure is different from that of geldanamycin, also blocked iNOS mRNA transcription. These cell biology findings were confirmed in infarcted myocardium where iNOS expression was markedly attenuated by Hsp90 inhibition in vivo. Intriguingly, further analyses showed that inhibiting Hsp90 had no significant effect on the activation of either IKK-NF-kappa B or JAK-STAT1 in LPS/IFN-gamma-stimulated cells. Neither was the nuclear transport of active NF-kappa B or STAT1 affected by Hsp90 inhibition. But Hsp90 inhibition markedly reduced the binding of active NF-kappa B and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was essential for NF-kappa B and STAT1 bindings to iNOS promoters inside cells. These studies reveal that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may represent a new approach to intervene iNOS expression in diseases.