Klf4 Alleviates Lipopolysaccharide-Induced Inflammation by Inducing Expression of MCP-1 Induced Protein 1 to Deubiquitinate TRAF6

Abstract

Background/Aims: Inflammation is an essential component of innate immunity against pathogens, but is tightly regulated, such as by Krüppel-like factor 4 (Klf4), to prevent injury. Klf4 also regulates macrophage polarization, although the mechanisms underlying both functions are poorly understood. The aim of this study was to investigate whether and how Klf4 prevents unregulated inflammation. Methods: The bone marrow-derived macrophages and RAW264.7 cell line were used. Quantitative real-time PCR was used to determine inflammatory cytokines (IL-1β, TNF-α, IL-6 and MCP-1), Klf4 and MCPIP1 transcript levels. Extraction of nuclear and cytoplasmic proteins and Immunoblotting were used to determine Klf4, MCPIP1, relative kinases from NF-κB pathway and K63-linked polyubiquitins expression in nucleus and cytoplasm separately. Dual luciferase reporter assay was used to analyze whether Klf4 mediate MCPIP1 transcription. Immunoprecipitation was used to determine the protein interaction among Klf4, MCPIP1, TRAF6 and K63-linked polyubiquitins. Secretion of IL-1β and TNF-α into sera in mice was measured by Enzyme-linked immunosorbent assay. Results: We found that exposure to lipopolysaccharides suppresses Klf4 expression, even as it induces release of pro-inflammatory cytokines. Strikingly, Klf4 overexpression significantly lowered cytokine secretion and NF-κB signaling in the cytoplasm following exposure to lipopolysaccharide, even though Klf4 was exclusively nuclear. The cytoplasmic effects are likely mediated by MCP-1 induced protein 1 (MCPIP1), a deubiquitinase and a key modulator of inflammation that accumulates both in the nucleus and cytoplasm in response to Klf4. Indeed, binding between MCPIP1 and K63 polyubiquitins is attenuated in macrophages overexpressing Klf4, suggesting that MCPIP1 is an intermediator induced by Klf4 in the nucleus to remove K63 polyubiquitins from TRAF6 in the cytoplasm, and thereby impede NF-κB and inflammatory signaling. Importantly, Klf4 overexpression in mice alleviated sepsis symptoms following exposure to lipopolysaccharides. Conclusion: The data highlight Klf4 as an essential MCPIP1-dependent modulator of innate immunity that protects against excessive and self-destructive inflammation.

The Role of Deubiquitinating Enzymes in Acute Lung Injury and Acute Respiratory Distress Syndrome

“…Acting as a deubiquitinating enzyme, MCPIP1 inhibits NF-κB and c-Jun N-terminal kinase (JNK) signaling pathways by removing the ubiquitin moieties from TNF receptor-associated factors (TRAFs), including TRAF2, TRAF3 and TRAF6 [150].As an RNase, MCPIP also regulates inflammatory cytokines like IL-6 by regulating RNA decay [151] and innate defense via degrading viral RNA [152]. MCPIP deubiquitinates TRAF6 to impede NF-κB signaling [89].…”

S-nitrosoglutathione protects lipopolysaccharide-induced acute kidney injury by inhibiting toll-like receptor 4–nuclear factor-κB signal pathway

“…Studies showed that the TLR4-NF-jB signalling pathway plays a key role in the production of inflammatory cytokines. [4][5][6] To investigate the regulatory mechanisms by which SNG inhibits LPS-induced inflammatory cytokine production in HK-2 cells, we incubated HK-2 cells in medium with different concentrations of SNG in vitro and then used LPS to induce cells to produce inflammatory cytokines. We detected the expression levels of TLR4-NF-jBassociated protein by Western blotting.…”

“…Previous studies reported that the incidence of septic AKI is 30–50% in the intensive care unit (ICU), and resulting in a mortality rate of 35–60% . The endotoxin produced by Gram‐negative bacteria is the main trigger for inflammatory response to sepsis, and lipopolysaccharide (LPS) as one of the major outer membrane components of Gram‐negative bacteria is the most important factor in the induction of septic AKI …”

“…[2,3] The endotoxin produced by Gram-negative bacteria is the main trigger for inflammatory response to sepsis, and lipopolysaccharide (LPS) as one of the major outer membrane components of Gramnegative bacteria is the most important factor in the induction of septic AKI. [4,5] Lipopolysaccharide first activates the Toll-like receptor 4 (TLR4) signalling pathway and further activates the downstream nuclear factor-jB (NF-jB) to induce cell apoptosis. [6] Therefore, the use of novel drugs to inhibit the TLR4-NF-jB signalling pathway may prevent AKI induced by LPS, thereby improving the prognosis of patients with sepsis.…”

MCPIP1 ribonuclease can bind and cleave AURKA mRNA in MYCN -amplified neuroblastoma cells

“…Additionally, the protein was also found to be a part of the deubiquitinase complex that facilitates the deubiquitination of tumour necrosis factor receptor-associated factor (TRAF) proteins. Deubiquitinase-associated activity of MCPIP1 leads to inhibition of nuclear factor kappa B (NFκB) signalling in immune cells [12][13][14].…”