Peroxisome proliferator–activated receptor γ (PPARγ) induces the gene expression of integrin α_Vβ₅ 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.

Introduction

Macrophages are markedly heterogenous cells that are responsive to various stimuli in either acute (infection) or chronic (metabolic syndrome) state (1, 2). M1 macrophages, activated upon classical pro-inflammatory cytokines (Toll-like receptor ligands and interferon-γ), are involved in Th1 immune response (3). In the presence of Th2 cytokines (interleukin-4, interleukin-13, and colony-stimulating factor), monocytes undergo an alternative M2 activation, characterized by an increased expression of Ym1, Arg1, Fizz1, and interleukin-10 (IL-10)² (4). Macrophage polarization is tightly regulated at the transcription level (5, 6). Interferon-regulatory factor 5 and signal transducer and activator of transcription 1 (STAT1) promote M1 polarization, whereas PPARγ cooperates with STAT6 to drive to a M2 phenotype (7).

PPARγ is a member of the nuclear receptor superfamily and a key regulator of lipid metabolism. It has been shown to suppress inflammation by inhibiting nuclear factor κ light-chain enhancer of activated B cells, STAT, and activator protein-1 pathways (8, 9). Constitutive expression of PPARγ in macrophages leads to an anti-inflammatory M2-like phenotype in adipose tissue. In contrast, knockdown of PPARγ impairs M2 polarization and insulin signaling (10). It has been demonstrated that PPARγ-dependent monocyte differentiation into M2 macrophage is beneficial to human carotid atherosclerosis (11). PPARγ activation by pioglitazone regulates M2 macrophage infiltration and stabilizes the neovessels in the infarct border zone, leading to a better outcome for patients after stroke (12). Previous studies showed that Arg1 and IL-10 are regulated by PPARγ and involved in M2 polarization (10, 13). However, the PPARγ-target genes driving the transcriptional program toward M2 polarization remain largely unknown.

Integrins are a family of ubiquitous transmembrane receptor expressed on a variety of cell types. Mammalian integrins comprise 18 α and 8 β subunits that constitute 24 heterodimers. Ligation of α and β subunits leads to a conformational change according to the extracellular ligands presented on the cell surface (14). In macrophages, integrin α_Mβ₂ (15), α_Dβ₂ (16), and α₅β₁ (17) have been implicated in phagocytosis, M1 polarization, and inflammasome activation. Integrin α₄ has recently been found to modulate metabolic inflammation in obesity (18). The absence of integrin β₃ favors macrophage polarization into M2a phenotype, which in turn increases fibrosis and impairs muscle regeneration (19). Mice lacking integrin β₃ in macrophage lineage cells have enhanced melanoma and breast cancer growth, because of increased tumor-promoting M2 macrophages (20). In addition, ligation of integrin α_Vβ₃ prevents macrophage differentiating into foam cells (21). In this study, we identified integrins α_V and β₅ as PPARγ target genes, and the heterodimer of integrin α_Vβ₅ was involved in M2 polarization.

Results

PPARγ regulated the expression of integrin α_Vβ₅

To investigate the activation of PPARγ on different integrins, RAW264.7 cells were treated with rosiglitazone, a PPARγ agonist for 24 h, the mRNA level of integrins α_M, α_V, α₅, α₆, α_L, α_D, α_IIb, β₁, β₂, β₃, and β₅ was detected with RT–qPCR. Integrins α_V, β₅, and α_M were increased upon rosiglitazone treatment, whereas integrins α₅, α₆, α_L, α_IIb, and β₁ remained unchanged. Integrin β₂ was down-regulated. Integrin β₃ was undetectable (Fig. S1).

Integrins α_V and β₅ are the most prominently up-regulated subunits and could be potentially ligated, prompting us to evaluate their precise regulations by PPARγ. To this end, RAW264.7 cells were treated with rosiglitazone for the indicated times or with different doses. Rosiglitazone increased the expression of integrins α_V and β₅ in a time- (Fig. 1, A and B) and concentration-dependent manner (Fig. 1, C and D). Moreover, they were up-regulated by pioglitazone, another agonist of PPARγ (Fig. 1, E and F). Selective PPARγ antagonists GW9662 abolished the stimulatory effect of rosiglitazone on integrin α_V and β₅ expressions (Fig. 1, G and H), indicating that the up-regulation by rosiglitazone was PPARγ-specific. Experiments conducted in human peripheral blood monocytes (hPBMCs) confirmed that both integrins α_V and β₅ could be induced by rosiglitazone and pioglitazone (Fig. 1, I and J). This induction was attenuated by GW9662 (Fig. 1, K and L). Taken together, these data suggested that both integrins α_V and β₅ could be induced by PPARγ activation. We then tested the expression of integrins α_V and β₅ in IL-4-promoted M2 macrophages. We found that both integrins α_V and β₅ could be induced by IL-4 in RAW264.7 cells (Fig. 2, A and B) and hPBMCs (Fig. 2C), demonstrating that integrins α_V and β₅ might be downstream molecules during M2 polarization.

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).

The heterodimer of integrin α_Mβ₂ was shown to mediate the inflammatory response in macrophages. To verify whether PPARγ activation could affect the expression of integrins α_M and β₂, RAW264.7 cells were treated with rosiglitazone as mentioned above. Expression of integrin α_M was increased in a time- (Fig. 3A) and dose-dependent manner (Fig. 3B) by rosiglitazone treatment, whereas integrin β₂ expression was significantly decreased (Fig. 3, C and D). Taken together, these results indicated that PPARγ was capable of regulating the expression of integrins α_M and β₂ in macrophages.

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).

Integrins α_V and β₅ were direct target genes of PPARγ

To investigate whether murine integrin α_V could be targeted by PPARγ, sequence analysis was performed using PPAR GENE (22) and JASPAR Database (http://jaspar.genereg.net/).³ Integrin α_V possesses two potential PPREs within the 2300-bp region upstream of murine integrin α_V gene (Fig. 4A). ChIP assay was executed to examine the bindings for PPARγ to the promoter region of integrin α_V. PPARγ could directly bind to integrin α_V promoter at either mPPRE-α_V1 (−1297/−1283) or mPPRE-α_V2 (−802/−788). The binding for mPPRE-α_V1 was increased with the treatment of rosiglitazone, whereas the binding for mPPRE-α_V2 remained unchanged (Fig. 4B). We then created two plasmid constructs containing different upstream regions of the integrin α_V promoter fused to the luciferase reporter. HEK 293 cells were treated with rosiglitazone after transfection with mLuc-α_V (containing mPPRE-α_V1 and mPPRE-α_V2) or mLuc-α_V-Δ (containing mPPRE-α_V2) plasmid. Luciferase activity of mLuc-α_V, but not mLuc-α_V-Δ, was increased by rosiglitazone treatment (Fig. 4C), suggesting that mPPRE-α_V1 could mediate the induction of integrin α_V gene by PPARγ. To further confirm the role of PPARγ in integrin α_V induction, mLuc-α_V vector was transfected into HEK 293 cells and then treated with rosiglitazone in the presence of a PPARγ specific antagonist GW9662 (Fig. 4D). The increase of luciferase activity of mLuc-α_V by rosiglitazone were prevented by GW9662, indicating that integrin α_V might be a direct target gene of PPARγ.

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.

Similarly, the promoter region of integrin β₅ possesses three PPREs (Fig. 4E). Both mPPRE-β₅1 (−1278/−1264) and mPPRE-β₅2 (−287/−273) could be bound to PPARγ, and these bindings were enhanced by rosiglitazone treatment. PPARγ did not bind to mPPRE-β₅3 (−240/−226) (Fig. 4F). Next, mLuc-β₅ (containing mPPRE-β₅1, mPPRE-β₅2, and mPPRE-β₅3), mLuc-β₅-Δ (containing mPPRE-β₅2 and mPPRE-β₅3), or mLuc-β₅-Δ′ (containing PPRE-β₅3) plasmids were transfected into HEK293 cells. Luciferase activity of mLuc-β₅, but not mLuc-β₅-Δ or mLuc-β₅-Δ′, was increased by rosiglitazone treatment (Fig. 4G), suggesting that mPPRE-β₅1 could mediate the induction of integrin β₅ gene by rosiglitazone. Meanwhile, the increase of luciferase activity of mLuc-β₅ by rosiglitazone was prevented by GW9662 (Fig. 4H), indicating that integrin β₅ might be direct target gene of PPARγ as well.

In addition, we also identified cognate PPARγ motifs in the regulation region of the human integrin α_V and β₅ genes. By using ChIP assay, we confirmed that PPARγ could bind to the promoter regions for human integrin α_V at hPPRE-α_V1 (−2109/−2095) or hPPRE-α_V2 (−1247/−1233) (Fig. 4I). Moreover, either the binding for hPPRE-α_V1 or for hPPRE-α_V2 was increased by rosiglitazone (Fig. 4J). Similarly, the functionality of PPRE in the human integrin β₅ gene was also confirmed (Fig. 4K).

Ligation of integrin α_Vβ₅ were increased by PPARγ activation

Given that integrins are obligate heterodimers, immunoprecipitation analysis was performed to examine the effect of rosiglitazone on α_Vβ₅ ligation. Consistent with the up-regulation of integrin α_V and β₅ expression by rosiglitazone, their ligation was significantly enhanced in RAW264.7 cells and hPBMCs (Fig. 5, A and B). Although integrin α_M was slightly increased, integrin α_Mβ₂ ligation was attenuated when treated with rosiglitazone in RAW264.7 cells and hPBMCs (Fig. 5, C and D). These results suggested that PPARγ activation might shift the ligation of different integrins, leading to a distinct downstream signaling pathway in macrophages.

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.

Integrin α_Vβ₅-mediated rosiglitazone-induced M2 macrophage polarization

To investigate the participation of integrin α_Vβ₅ in PPARγ-induced M2 polarization, siRNA strategy was first used following a treatment with rosiglitazone. Rosiglitazone induced mRNA and protein levels of M2 marker genes, including Ym1, Arg1, and Fizz1. Importantly, either siRNA against integrin α_V or β₅ attenuated the induction of M2 marker genes by rosiglitazone. Combination of siRNA against integrins α_V and β₅ abrogated this augmentation in RAW264.7 cells (Fig. 6, A and B) and hPBMCs (Fig. 6C). Rosiglitazone exhibited an anti-inflammatory effect in monocytes/macrophages by reducing the expression of M1 marker genes such as iNOS, TNFα, and IL-6. Neither siRNA against integrin α_V/β₅ nor their combination could reverse the reduced expression of iNOS, TNFα and IL-6 expressions (Fig. S2, A and B).

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).

Furthermore, pharmacological blockage with cilengitide, a potent inhibitor blocking the accessibility of integrin α_Vβ₅ to their ligands (23), effectively abolished rosiglitazone-induced Ym1, Arg1, and Fizz1 in RAW264.7 cells (Fig. 7, A and B) and hPBMCs (Fig. 7C). In contrast, cilengitide had no effect on rosiglitazone-decreased expression of M1 marker genes (Fig. S3, A and B). These data suggested that the heterodimer of integrin α_Vβ₅ was required in PPARγ-induced M2 polarization.

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.

Discussion

In this present study, we demonstrated a novel mechanism by which PPARγ regulates macrophage polarization via integrin α_Vβ₅ induction. These results also provided evidence that a specific integrin heterodimer plays an important role in M2 polarization.

Integrins are important signaling receptors that mediate the interactions of the cells with extracellular matrix (24). They are involved in multiple inflammatory responses, including coronary atherosclerosis, obesity, etc. (25, 26). However, the gene regulation of specific integrin subunits in macrophages has not been well-characterized. Here we showed that PPARγ transcriptionally activated integrins α_V and β₅. GW9662, an antagonist of PPARγ, attenuated the up-regulation of integrins α_V and β₅ by rosiglitazone. It is noticed that GW9662 elevated the basal level of integrins α_V and β₅ by an unrecognized mechanism, which has been reported in the case of the other nuclear receptor antagonist (27). We identified murine PPRE-α_V1 (−1297/−1283) and murine PPRE-β₅1 (−1278/−1264) as functional binding sites to trigger integrin α_V and β₅ transcription, respectively. Meanwhile, we found that the expression of inflammatory integrins α_M and β₂ was regulated by PPARγ as well.

Integrin is strictly heterodimer of α and β subunits, which are ligated by extracellular stimuli and required for its signaling transduction (24). The mechanism of the formation of integrin heterodimer has not been well-understood. The heterodimer of integrin α₂β₁ ligated by C1q-containing immune complexes is required for peritoneal mast cells activation during innate immunity (28). Adhesion of monocytes to the endothelium results in integrin α_Vβ₃ ligation and prevents macrophage transition into foam cells (21). Here, we found that a signaling pathway initiated by integrin α_Vβ₅ ligation could participate in macrophage M2 polarization. Meanwhile, ligation of integrin α_Mβ₂, which has been implicated in inflammatory response (26), was decreased upon PPARγ activation. This is the first evidence that PPARγ switches the ligation of specific integrin subunits, leading to distinct cellular functions in macrophages.

PPARγ has been shown to function as insulin sensitizers and thus improve hyperglycemia in patients with type 2 diabetic mellitus (29). Knockdown of PPARγ in immune cells reduces insulin sensitivity by decreasing the infiltration of macrophages into white adipose tissue (30). Dominant mutations in PPARγ cause insulin resistance accompanied by early onset of severe hypertension (31, 32). The aortas from troglitazone-treated mice show decreased accumulation of macrophages in atherosclerotic lesions and attenuated expression of numerous inflammatory markers such as TNFα and iNOS (33), which is consistent with our results that the expression and ligation of inflammatory integrin α_Mβ₂ were decreased by PPARγ activation in macrophages. Alternation of macrophage polarization and function requires precise regulation of master factors, including cytokines (IL-4 and IL-13) and transcription factors like PPARγ. PPARγ activates anti-inflammatory gene expressions, such as Arg-1 and IL-10 through the PPREs at their promoter regions (10, 13). In this study, we found that integrins α_V and β₅ were PPARγ target genes and necessary to PPARγ-induced M2 polarization. To the best of our knowledge, this is the first evidence implicating a specific integrin heterodimer in M2 macrophages.

Integrins are transmembrane receptors that respond to extracellular stimuli. Ligation of two integrin subunits is not sufficient to induce the downstream signaling; thus it is important to identify the ligands of integrin α_Vβ₅ during M2 polarization, especially on the basis of physiopathological context. Integrin α_Vβ₅ binds to a variety of extracellular matrix proteins, including osteopontin, fibronectin, vitronection, von Willebrand factor, and thrombospondin. An in vitro study demonstrated that IL-10 acts synergistically with IL-18 to amplify the production of osteopontin, thereby augmenting M2 polarization of macrophage (34). The osteopontin-generated M2 macrophages exhibit a protective effect in vascular calcification of patients with hypertension (35). Although the culture of macrophage on fibronectin-coated surface does not induce a M2 phenotype (36), the production and deposition of fibronectin are common features during M2 polarization (37, 38), which is believed to govern the remodeling process after tissue damage (39). Moreover, the downstream signaling of integrin α_Vβ₅ in macrophages should be implemented in our further studies. It has been previously shown that the selective inhibitor of Rok2 (Rho-associated kinase 2) decreases M2-like macrophages (40). As an upstream receptor of Rho, integrin α_Vβ₅ activation on Rok2 could explain how it participated in M2 polarization. Furthermore, because macrophages exhibit a longer shape when differentiated into M2 phenotypes (41), the effect of integrin α_Vβ₅ on cytoskeleton rearrangement (42) could be another hypothesis to elucidate the involving mechanism. Taken together, our results suggested a novel molecular mechanism with which the induction and ligation of integrin α_Vβ₅ participate in PPARγ-induced M2 polarization, whereas PPARγ represses the inflammation by targeting the expression and the ligation of integrin α_Mβ₂ (Fig. 8), which may provide a potent target against inflammatory diseases.

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.

Experimental procedures

Reagents

Rosiglitazone, GW9662, and recombinant IL-4 were from Sigma–Aldrich. Pioglitazone and cilengitide were from Selleck Chemicals (Houston, TX). Antibodies against Arg1, β-actin, integrin α_M, and integrin β₂ were from Santa Cruz Biotechnology (Dallas, TX). Antibodies against integrins α_V and β₅ were from Cell Signaling (Danvers, MA). Antibodies against Ym1 and Fizz1 were from Abcam (Cambridge, MA).

Cell culture, isolation, and transfection

Murine monocytic cell line RAW264.7 and HEK 293 cells were obtained from ATCC (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37 °C. Human PBMCs were isolated from healthy donors by Ficoll–Hypaque density centrifugation. After washing three times, hPBMCs were incubated in RPMI 1640 medium supplemented with 10% fetal bovine for 4 days. The experiments were approved by the institutional ethics review board of Xi'an Jiaotong University (approval XJTULAC2018-497) and performed in accordance with the National Institutes of Health guidelines.

RNA extraction and reverse transcriptase–PCR (RT–qPCR)

Total RNA was extracted from RAW264.7 cells and hPBMCs by using TRIzol (Invitrogen). Quantitative RT–qPCR was performed using the SYBR Green technique (Promega, Madison, WI). Primer sequences were described in Table S1. GAPDH was used as a housekeeping gene.

Plasmids and luciferase reporter assay

mLuc-α_V (−1383/+109), mLuc-α_V-Δ (−831/+109), Luc-β₅ (−1346/+40), mLuc-β₅-Δ (−443/+40), and mLuc-β₅-Δ′ (−270/+40) plasmids were made by PCR cloning into the pGL3 basic luciferase-reporter plasmid. The cells were co-transfected with a reporter gene and a β-gal plasmid. Luciferase and the β-gal activities were measured as previously described (43).

ChIP assay

The cells were cross-linked with 0.75% formaldehyde before harvesting. Sheared chromatin was immunoprecipitated with an anti-PPARγ antibody (or control IgG) and pulled down with protein A/G–Sepharose beads (Santa Cruz). The eluted immunoprecipitates were digested with proteinase K to reverse the cross-link between DNA and proteins. DNA was extracted and subjected to PCR experiment with specific primers flanking the putative PPARγ binding motifs. Primer sequences were described in Table S2.

Immunoblotting and immunoprecipitation

Proteins were extracted in RIPA buffer supplemented with protease inhibitors. Protein concentrations were measured using the BCA protein assay. Immunoblotting was performed with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies followed by ECL detection. Immunoblots shown were representative of three independent experiments.

For immunoprecipitation, cell lysates were incubated with the appropriate antibodies or control IgG at 4 °C overnight followed by incubation with protein A/G-Sepharose beads. Immunoprecipitates were washed with NETN buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, and 0.5% NP-40). Immunoblots shown were representative of three independent experiments.

siRNA and transfection

RAW264.7 cells or hPBMCs were transfected with adequate species of integrin α_V, integrin β₅, or scrambled siRNA (si Ctrl). Sequences were described in Table S3. Experiments using these cells were executed at 24 h after transfection.

Statistical analysis

The results are reported as means ± S.D. Comparisons within and between groups were performed using analysis of variance and Mann–Whitney U test, respectively. p < 0.05 was considered significant.

Author contributions

Q. Y. and N. W. conceptualization; Q. Y., J. L., Z. Z., C. Z., and N. W. data curation; Q. Y. and Z. Z. formal analysis; Q. Y. and N. W. supervision; Q. Y., J. L., L. X., and N. W. funding acquisition; Q. Y., J. L., Z. Z., and N. W. validation; Q. Y., J. L., F.L., C. Z., and N. W. investigation; Q. Y. and N. W. visualization; Q. Y., J. L., F.L., C. Z., B.L., L. X., and N. W. methodology; Q. Y. and N. W. writing-original draft; Q. Y. and N. W. writing-review and editing; N. W. resources.