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

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Abstract
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.
Funding Information
  • National Heart, Lung, and Blood Institute (R01-HL081860)
  • U.S. Department of War (W81XWH-15-1-0624)
  • U.S. Department of War (W81XWH-20-1-0688)

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