Downregulation of Caveolin‐2 Is a Novel Mechanism Involved in Activation of Inducible Nitric Oxide Synthase (iNOS) in Mouse Peritoneal Macrophages

Here we report that membrane protein caveolin-2 (Cav-2) is regulated during activation of primary mouse peritoneal macrophages and RAW264.7 macrophage cell line by a bacterial lipopolysaccharide (LPS) and a cytokine, interferon gamma (IFN-γ). We also show that downregulation or loss of Cav-2 increases expression of inducible nitric oxide (NO) synthase (iNOS) and subsequent NO production in activated macrophages. Treatment with LPS and IFN-γ downregulated Cav-2 at the protein and mRNA level. Moreover, LPS- and IFN-γ-induced downregulation of Cav-2 inversely correlated with iNOS expression levels. Mechanistically, activation of NF-κB pathway was responsible for downregulation of Cav-2 by LPS and IFN-γ since pharmacological inhibition of NF-κB activation with pyrrolidine dithiocarbamate (PDTC) prevented Cav-2 downregulation and iNOS activation. To test if LPS- and IFN-γ-induced downregulation of Cav-2 could possibly be involved in macrophage activation, we used a combination of siRNA knockdown and genetic deletion/knockout (KO) approaches. Remarkably, reduction of Cav-2 using siRNA approach resulted in enhanced STAT1 phosphorylation, iNOS expression, and increased NO production in LPS- and LPS plus IFN-γ-stimulated macrophages. Conversely, genetic deletion of Cav-2 robustly enhanced IFN-γ-stimulated iNOS expression and subsequent NO production. Overall, our data suggest that Cav-2 is not only regulated during macrophage activation, but it also may be an important physiological regulator of macrophage activation via preventing excessive STAT1 signaling and iNOS stimulation.