Peroxisome proliferator-activated receptor γ (PPARγ) induces the gene expression of integrin αVβ5 to promote macrophage M2 polarization

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily and polarizes the macrophages into an anti-inflammatory M2 state. Integrins are transmembrane receptors that drive various cellular functions, including monocyte adhesion and foam cell formation. In this study, we first reported that the expression of integrins α_V and β₅ was up-regulated by PPARγ activation in RAW264.7 cells and human peripheral blood monocytes. Luciferase reporter and ChIP assay revealed that PPARγ directly bound to the potential PPAR-responsive elements sites in the 5'-flanking regions of both murine and human integrin α_V and β₅ genes, respectively. In addition, we showed that PPARγ augmented the ligation of integrins α_V and β₅ Knockdown of integrin α_Vβ₅ by siRNA strategy or treatment with cilengitide, a potent inhibitor of integrin α_Vβ₅, attenuated PPARγ-induced expression of Ym1 (chitinase-like protein 3), Arg1 (Arginase1), Fizz1 (resistin-like molecule RELMα), and other M2 marker genes, suggesting that the heterodimers of integrin α_Vβ₅ were involved in PPARγ-induced M2 polarization. In conclusion, these results provided novel evidence that PPARγ-mediated gene expression and the ensuing ligation of integrins α_V and β₅ are implicated in macrophage M2 polarization.

Keywords: gene regulation; inflammation; integrin; integrin alpha V; integrin beta 5; macrophage polarization; peroxisome proliferator-activated receptor (PPAR); protein-protein interaction.

Figure 1.

Expression of integrins α_V and β₅ was increased by PPARγ activation. A–D, RAW264.7 cells were incubated with 10 μmol/liter rosiglitazone (RGZ) for the indicated time (A and B) or with the indicated concentrations of RGZ for 24 h (C and D). Cell lysates were analyzed for the level of integrins α_V and β₅ by using RT–qPCR or immunoblotting. *, p < 0.05 versus control (Ctrl; integrin α_V); #, p < 0.05 versus Ctrl (integrin β₅). E–J, RAW264.7 cells and hPBMCs were stimulated with RGZ (10 μmol/liter), pioglitazone (PGZ, 10 μmol/liter), or both. The integrin α_V and β₅ mRNA (E and I) or protein (F and J) levels were examined by RT–qPCR or immunoblotting. G–L, cells were pretreated with or without GW9662 for 1 h and then exposed to RGZ (10 μmol/liter) for 24 h. Cell lysates were analyzed to determine the mRNA (G and K) and protein (H and L) levels of integrin α_V and β₅. *, p < 0.05.

Figure 2.

Expression of integrins α_V and β₅ in IL-4–induced M2 macrophages. RAW264.7 cells and hPBMCs were stimulated with IL-4 (10 ng/ml) for 24 h. Cell lysates were analyzed for the levels of integrins α_V and β₅ using RT–qPCR (A and C) or immunoblotting (B). *, p < 0.05 versus control (Ctrl; integrin α_V); #, p < 0.05 versus control (integrin β₅).

Figure 3.

Expression of integrins α_M and β₂ was modulated by PPARγ activation. RAW264.7 cells were incubated with 10 μmol/liter RGZ for the indicated times (A and C) or with indicated concentrations of RGZ for 24 h (B and D). The cell lysates were analyzed for the level of integrin α_M and β₂ by using RT–qPCR or immunoblotting. Immunoblots of A and C were from the same representative experiment consecutively used to detect integrin α_M, integrin β₂, and β-actin. The results were separately presented, but the loading control (β-actin) was identical. The same situation applies to B and D. *, p < 0.05 versus control (Ctrl).

Figure 4.

PPARγ bound and activated the integrin α_V and β₅ promoters. Potential PPREs located in the regions of murine integrin α_V (A) and β₅ (E) promoters were schematically presented. RAW264.7 cells were treated with RGZ (10 μmol/liter). The indicated murine PPREs of integrin α_V (B) or β₅ (F) were analyzed by ChIP assay with the use of anti-PPARγ antibody. IgG was used as an isotype control (Ctrl). HEK 293 cells were transfected with a serial of pGL3 basic vectors in which different fragments of integrin α_V (C, mLuc-α_V and mLuc-α_V-Δ) or β₅ (G, mLuc-β₅, mLuc-β₅-Δ, and mLuc-β₅-Δ′) promoter have been cloned. After treatment with RGZ (10 μmol/liter) for 24 h, luciferase activity was measured and normalized to that of β-gal. mLuc-α_V (D) or mLuc-β₅ (H) transfected HEK293 cells were incubated with RGZ (10 μmol/liter) after pretreatment with GW9662 (20 μmol/liter). Luciferase activity was measured and normalized to that of β-gal. Schematic presentation of PPREs located in the regions of human integrin α_V and β₅ (I) promoters. hPBMCs were treated with RGZ (10 μmol/liter). The indicated human PPREs of integrin α_V (J) or β₅ (K) were analyzed by ChIP assay with the use of anti-PPARγ antibody. IgG was used as an isotype control. *, p < 0.05.

Figure 5.

Rosiglitazone increased the formation of integrin α_Vβ₅ heterodimer. RAW264.7 cells and hPBMCs were treated with RGZ for 24 h. Immunoblotting of anti-integrin α_V (A and B) or anti-integrin β₂ (C and D) immunoprecipitates were performed. IgG was used as a negative control. Ctrl, control; IP, immunoprecipitation.

Figure 6.

Integrin α_Vβ₅ knockdown abolished rosiglitazone-promoted M2 polarization. RAW264.7 cells and hPBMCs were transfected with murine and human si control (Ctrl), si α_V, si β₅ or si α_V+β₅, respectively. Then the cells were treated with RGZ (10 μmol/liter) for 24 h. The cell lysates were analyzed for the level of Ym1, Arg1, and Fizz1 by using RT–qPCR (A and C) or immunoblotting (B).

Figure 7.

Cilengitide partially inhibited rosiglitazone-induced M2 polarization. RAW264.7 cells and hPBMCs were pretreated with cilengitide (Cilen, 1μmol/liter) for 30 min and then incubated with RGZ (10 μmol/liter) for 24 h. Lysates were analyzed for the level of Ym1, Arg1, and Fizz1 by using RT–qPCR (A and C) or immunoblotting (B). Ctrl, control.

Figure 8.

Mechanism of integrin in PPARγ-induced M2 macrophage polarization. PPARγ activates the expression of integrin α_V and β₅ by targeting the PPREs in their promoter regions. The formation of integrin α_Vβ₅ heterodimer is therewith increased, leading to M2 macrophages polarization. Meanwhile, PPARγ activation regulates the expression and the ligation of integrin α_M and β₂, leading to a reduced inflammatory phenotype in macrophages.