Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays critical roles in inflammation and host defense. iNOS expression is induced by inflammatory stimuli and exhibits constant activity once expressed. Hence NO production from iNOS has been thought to be primarily controlled via enzyme expression. In this study, we identify an array of novel mechanisms showing that iNOS is regulated at multiple levels including gene expression, protein aggregation, and protein degradation.
Calmodulin (CaM) has always been thought as an iNOS cofactor facilitating electron transfer amid NO syntheses. We now show that CaM is essential for iNOS induction. CaM inhibition or knockdown prevented iNOS expressions in macrophages stimulated by inflammatory mediators. Further studies revealed that CaM acted through Ca2+/CaM-dependent kinase II (CaMKII), which functioned as a cardinal initiator in iNOS gene transactivation via both LPS-NF-¿¿¿¿¿B and IFN-γ-STAT1 pathways. Our study also found that iNOS gene transactivation required cytosolic Ca2+ elevations. CaM or CaMKII inhibition was found to prevent iNOS induction in endotoxemic mice and improved survival rates. These studies extend the role of CaM from an enzyme cofactor to an essential modulator in iNOS gene transactivation.
Once expressed, biological iNOS output is determined by the levels of functional enzyme, which is influenced by protein stability. Our studies found that while initially existing as a soluble protein, iNOS progressively formed inactive protein aggregates. Blocking NO production prevented iNOS aggregation inside cells. iNOS aggresome formation could be recaptured by exposing cells to exogenous NO. The finding that NO per se induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.
After elucidating NO-mediated iNOS aggregation in physiological conditions, we then investigated iNOS protein stability under pathological circumstances upon heat shock protein 90 (Hsp90) or CaM inhibition. Hsp90 was previously reported to play an important role in iNOS function and gene expression. We now find that dissociation of Hsp90 from iNOS led to iNOS aggregation and these aggregates were cleared by ubiquitin-proteasome system. Further studies revealed that the SPRY domain-containing SOCS box protein 2 (SPSB2), but not the C-terminus of heat shock protein 70-interacting protein (CHIP), was essential for aggregated iNOS turnover in Hsp90-inhibited cells. Our studies found that CaM inhibition disrupted CaM-iNOS interaction, exposing the hydrophobic CaM-binding domain on iNOS which mediated iNOS aggregation. These studies identified novel roles of Hsp90 and CaM in modulating iNOS protein stability.
To gain a complete understanding of iNOS regulation, we investigated the ubiquitination site responsible for proteasomal degradation of iNOS. The N-terminal 1-100 amino acids were found to be essential for iNOS degradation. However, arginine replacement of all lysines within this region failed to prevent iNOS degradation. Further studies identified several non-lysine residues among the N-terminal region of iNOS crucial for its ubiquitination and proteasomal degradation.
In summary, our investigations change the notion that iNOS biology is largely dependent on the levels of its gene expression. The knowledge gained from this work provided a comprehensive understanding of iNOS regulation via three interrelated aspects including protein induction, stability and degradation.