E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression

Abstract

Stimulated macrophages generate potent anti-microbial reactive oxygen and nitrogen species within their phagosomes. Previous studies have shown that the E3 ubiquitin ligase natural killer lytic-associated molecule (NKLAM) is a macrophage phagosomal protein that plays a role in macrophage anti-bacterial activity. In vivo, NKLAM-knockout (KO) mice produce less nitric oxide (NO) upon exposure to lipopolysaccharide (LPS) than wild type (WT) mice. In vitro, we found that NO production and inducible nitric oxide synthase (iNOS) protein were diminished in LPS-stimulated KO bone marrow-derived and splenic macrophages. Additionally, LPS-stimulated NKLAM-KO macrophages displayed defects in STAT1 tyrosine phosphorylation and production of interferon beta (IFNβ). The JAK/STAT pathway is critical for the production of IFNβ, which augments iNOS protein expression in mice. iNOS protein expression is also regulated by the transcription factor NFκB, thus we investigated whether NKLAM influences NFκB function. LPS-stimulated NKLAM-KO macrophages showed evidence of delayed nuclear translocation of the NFκB subunit p65. This was associated with a reduction in p65/DNA colocalization. The defect in p65 translocation was independent of IKBα degradation. NKLAM-KO macrophages also expressed less p65 and showed evidence of defective p65 phosphorylation at serine 536. Importantly, LPS-stimulated NKLAM-KO macrophages have diminished NFκB transcriptional activity as assessed by transfection of a luciferase reporter plasmid. Collectively, our data implicate NKLAM as a novel modulator of macrophage iNOS expression.

E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity

“…6B, treatment with IFNγ stimulated WT iNOS mRNA expression that was significantly elevated over NKLAM-KO. These results are consistent with our data that showed NKLAM-KO BMDM express less iNOS in response to LPS [27]. The effect of NKLAM on STAT1-regulated genes was not limited to genes with GAS elements.…”

Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection

“…pneumoniae (SP) was significantly less in NKLAM-KO macrophages as compared to WT macrophages ( Fig 3C and 3D ). This observation is in line with our previous results that show NKLAM-KO BMDM express less iNOS in response to LPS than WT macrophages [ 12 ] and suggests a more global role for NKLAM in mediating iNOS expression. Collectively, these data show that NKLAM-KO mice experience a significant defect in leukocyte transmigration into the lung in the response to S .…”

“…There is an emerging body of evidence suggesting that RBR ubiquitin ligases are involved in regulating the expression of pro-inflammatory cytokines. Studies from our laboratory have shown that NKLAM is involved in enhancing the expression of IFNγ or RANTES/CCL5 in response to LPS and IFNγ, respectively [ 8 , 12 ]. The knockdown of E3 ubiquitin ligase Parkin with siRNA or the use of Parkin-KO mice demonstrated that Parkin, in part, regulates the expression of lung IL-6 and TNFα as well as the expression of IL-6 and IL-8 from endothelial cells [ 13 ].…”

Effects of Lycium barbarum polysaccharides with different molecular weights on function of RAW264.7 macrophages

“…To confirm these results, we also determined the iNOS mRNA expression in LBP3-treated RAW264.7 macrophages. The iNOS is an enzyme that can induce the synthesis of NO in macrophages (Lawrence, Gullickson, & Kornbluth, 2015). Once being activated, the macrophages express large amounts of iNOS, while in the resting state there is nearly no expression.…”