Possible regulation of Toll-like receptor 4 by lysine acetylation through LPCAT2 activity in RAW264.7 cells

Abstract

Inflammation is central to several diseases. TLR4 mediates inflammation by recognising and binding to bacterial lipopolysaccharides and interacting with other proteins in the TLR4 signalling pathway. Although there is extensive research on TLR4-mediated inflammation, there are gaps in understanding its mechanisms. Recently, TLR4 co-localised with LPCAT2, a lysophospholipid acetyltransferase. LPCAT2 is already known to influence lipopolysaccharide-induced inflammation; however, the mechanism of LPCAT2 influencing lipopolysaccharide-mediated inflammation is not understood. The present study combined computational analysis with biochemical analysis to investigate the influence of LPCAT2 on lysine acetylation in LPS-treated RAW264.7 cells. The results suggest for the first time that LPCAT2 influences lysine acetylation in LPS-treated RAW264.7 cells. Moreover, we detected acetylated lysine residues on TLR4. The present study lays a foundation for further research on the role of lysine acetylation on TLR4 signalling. Moreover, further research is required to characterise LPCAT2 as a protein acetyltransferase.

Keywords: acetylation/deacetylation; acetyltransferases; bioinformatics; immunomodulation; inflammation; toll-like receptors.

Figure 1. Effect of LPCAT2 knockdown on RAW264.7 cells

Transfection of RAW264.7 cells with LPCAT2 siRNA causes ≥70% decrease in LPCAT2 gene expression and ≥50% decrease in LPCAT2 protein expression (A) *P≤0.05. Fold change shows the optical density of LPCAT2 normalised to GAPDH. Data represent mean of at least three independent experiments (n≥3) ± standard error (A) of Western blots (B) Transfection of siRNA into RAW264.7 cells does not cause a significant difference in total RNA and total protein (C), and in the expression of housekeeping genes ATP5B and GAPDH (D) when compared with cells with no transfected siRNA. Data represent the median of at least three independent experiments (n≥3) ± interquartile range.

Figure 2. Analysis of the relatedness of LPCAT2 To other lysine acetyltransferases in mice

(A) Phylogenetic tree showing the degree of relatedness of LPCAT1 gene, LPCAT2 gene, and genes of other KATs. Numbers indicate node positions. (B–D) Sequence alignment of proteins in node 7, white space indicates gaps in alignment. Letters symbolising amino acids are IUPAC standards.

Figure 3. Analysis of pan-lysine acetylation in lipopolysaccharide-stimulated RAW264.7 cells

(A) Western blot image of lysine acetylated proteins. Acetylated α-tubulin was used as a positive control. Lane 1: Control siRNA, Lane 2: LPCAT2 siRNA, Lane 3: Control siRNA + LPS, Lane 4: LPCAT2 siRNA + LPS. (B) Fold change in optical density of lysine acetylated proteins normalised to acetylated α-tubulin. Mid-bars represent median, boxes represent interquartile range, upper bar represent maximum, and lower bar represent minimum. Welch’s T-test P values; No LPS versus LPS (P=0.03), No LPS versus negative siRNA (P=0.73), LPS versus LPCAT2 siRNA + LPS (P=0.002). Data were obtained from at least three independent experiments.

Figure 4. Detection of acetylated lysine on TLR4 in RAW264.7 cells

(A) Western blot of TLR4 in after immunoprecipitation of TLR4. Lane 1: Cleared cell lysate (No LPS treatment); Lane 2: Cleared cell lysate (LPS treatment); Lane 3: TLR4 eluate (no LPS treatment); Lane 4: TLR4 eluate (LPS treatment). (B) Western blot of acetylated lysine after immunoprecipitation of TLR4. Lane 1: TLR4 eluate (no LPS treatment); Lane 2: TLR4 eluate (LPS treatment); Lane 3: normal mouse IgG eluate (no LPS treatment); Lane 4: normal mouse IgG eluate (LPS treatment); Normal mouse IgG is an isotype control for immunoprecipitation. (C) Optical densities of acetylated lysine detected on TLR4. The optical densities were normalised to total TLR4 (optical density of TLR4 in cleared cell lysate + optical density of TLR4 in TLR4 Eluate). Welch’s T-test shows no significant difference between no LPS treatment and LPS treatment (P=0.99). Data are obtained from three independent experiments.

Figure 5. Effect of LPCAT2 knockdown on mRNA expression IFNβ and IP10

Knockdown of LPCAT2 affects the gene expression of IFNβ (A) and interferon-inducible protein 10, IP10 (B) and protein expression of IP10 (C) in RAW264.7 cells stimulated with TLR4 ligand (100 ng/ml of E. coli O111:B4 lipopolysaccharide). Non-treated RAW264.7 cells did not show any significant difference. Welch’s T-test (*P≤0.05). Data represent the mean of at least three independent experiments (n≥3) ± standard error.

Figure 6. Knockdown of LPCAT2 reduces detected acetylated lysine residues on TLR4

(A) Acetylated lysine blots (WB) of TLR4 precipitates (IP). Lane 1: No siRNA, Lane 2: Control siRNA, Lane 3: LPCAT2 siRNA. TLR4 dot blots of same quantity of proteins from TLR4 eluates were used as controls of protein amounts. (B) Optical densities of blots in (A). Data are obtained from one experiment.