PAK1 Silencing Attenuated Proinflammatory Macrophage Activation and Foam Cell Formation by Increasing PPAR γ Expression

Abstract

Macrophage polarization in response to environmental cues has emerged as an important event in the development of atherosclerosis. Compelling evidences suggest that P21-activated kinases 1 (PAK1) is involved in a wide variety of diseases. However, the potential role and mechanism of PAK1 in regulation of macrophage polarization remains to be elucidated. Here, we observed that PAK1 showed a dramatically increased expression in M1 macrophages but decreased expression in M2 macrophages by using a well-established in vitro model to study heterogeneity of macrophage polarization. Adenovirus-mediated loss-of-function approach demonstrated that PAK1 silencing induced an M2 macrophage phenotype-associated gene profiles but repressed the phenotypic markers related to M1 macrophage polarization. Additionally, dramatically decreased foam cell formation was found in PAK1 silencing-induced M2 macrophage activation which was accompanied with alternation of marker account for cholesterol efflux or influx from macrophage foam cells. Moderate results in lipid metabolism and foam cell formation were found in M1 macrophage activation mediated by AdshPAK1. Importantly, we presented mechanistic evidence that PAK1 knockdown promoted the expression of PPARγ, and the effect of macrophage activation regulated by PAK1 silencing was largely reversed when a PPARγ antagonist was utilized. Collectively, these findings reveal that PAK1 is an independent effector of macrophage polarization at least partially attributed to regulation of PPARγ expression, which suggested PAK1-PPARγ axis as a novel therapeutic strategy in atherosclerosis management.

Figure 1

PAK1 expression is induced in M1 but reduced in M2 macrophages. (a, b) PAK1 mRNA levels in PMs and BMDMs subjected to PBS, IL-4, or LPS stimulation for 24 hours. n = 3. (c, d) Western blot analysis of PAK1 protein levels in PMs and BMDMs administrated with PBS, IL-4, or LPS for 24 hours. n = 3. (e) Representative images of double immunofluorescence staining of BMDMs with an anti-PAK1 antibody (red) and macrophage marker (CD68, green) treated with PBS, IL-4, or LPS for 24 hours. The integral optical density of PAK1 was presented. Scale bar = 50 μm. n = 3. ^∗P < 0.05 versus PBS group, ^#P < 0.05 versus IL-4 group. PMs: peritoneal macrophages; BMDMs: bone marrow-derived macrophages; PBS: phosphate-buffered saline; IL-4: interleukin-4; LPS: lipopolysaccharides.

Figure 2

PAK1 promotes M2 polarized macrophages. (a, b) The PAK1 expression in BMDMs infected with AdshPAK1 or AdshRNA. n = 3. (c) mRNA expression levels of M2 macrophage markers in BMDMs by PAK1 knockdown with IL-4 stimulation. n = 3. (d) Protein expression levels of Arg-1 and IL-10 in BMDMs by PAK1 knockdown with IL-4 stimulation. n = 3. ^∗P < 0.05 compared with control group. Arg-1: arginase-1.

Figure 3

PAK1 attenuates the M1 polarized macrophages. (a–f) RT-PCR analysis of expression levels of M1 macrophage markers in BMDMs by PAK1 knockdown upon LPS treatment. n = 3. (g) Western blot analysis of TNF-α and IL-6 protein level in BMDMs by PAK1 knockdown upon LPS treatment. n = 3. ^#P < 0.05 compared with control group. TNF-α: tumor necrosis factor-α; iNOs: inducible no synthase.

Figure 4

Decreased foam cell formation by PAK1 silencing. (a) Oil-red staining of BMDMs infected with AdshPAK1 or AdshRNA, which previously stimulated with IL-4 or LPS and administrated with Ox-LDL. Scale bar = 100 μm. n = 6‐10. (b–f) Alternation of the expression of SR-A and CD36, and ABCA1 and ABCG1 in BMDMs infected with AdshPAK1, which previous stimulated with IL-4 or LPS and administrated with Ox-LDL at mRNA (b–e) and protein (f) levels, respectively. n = 3. ^∗P < 0.05 or ^#P < 0.05 compared with control group. SR-A: scavenger receptor type A; ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1 (ABCG1).

Figure 5

Upregulated PPARγ expression by PAK1 silencing. (a) mRNA levels of the representative regulators for macrophage polarization in BMDMs by PAK1 knockdown with IL-4 stimulation. n = 3. ^∗P < 0.05 compared with AdshRNA group. (b) Protein level of PPARγ in BMDMs by PAK1 knockdown with IL-4 or LPS stimulation. n = 3. ^∗P < 0.05 or ^#P < 0.05 compared with control group. (c–f). RT-PCR (c, d) and Western blot (e, f) analysis of M2 and M1 marker gene expression in macrophage transfected with AdshRNA or AdshPAK1 and treated with LPS or IL-4, which then cultured with G3335 or control. n = 3. ^∗P < 0.05 compared with AdshRNA group; ^#P < 0.05 vs. AdshPAK1 group; ^†P < 0.05 vs. AdshRNA with G3335 group. (g) Immunoblotting with PPARγ or PAK1antibody was performed on co-IP of PAK1 using PAK1 antibody. (h) Schematic diagram of the molecular mechanisms underlying PAK1-regulated macrophage polarization and foam cell. PPARγ: peroxisome proliferator-activated receptor γ.