Collagen XV Promotes ER Stress-Induced Inflammation through Activating Integrin β1/FAK Signaling Pathway and M1 Macrophage Polarization in Adipose Tissue

Abstract

Collagen XV (Col XV), a basement membrane (BM) component, is highly expressed in adipose tissue, and studies have found that Col XV is related to extracellular matrix (ECM) remodeling involving in adipose tissue fibrosis and inflammation. Furthermore, the ECM is essential for maintaining normal development and tissue function. In this study, we found that Col XV is related to the endoplasmic reticulum stress (ERS) and inflammation of adipose tissue. Moreover, we found that overexpression of Col XV in mice could cause macrophages to infiltrate white adipose tissue (iWAT). At the same time, the expression of the ERS sensor IRE1α (Inositol-Requiring Enzyme-1α) was significantly up-regulated, which intensified the inflammation of adipose tissue and the polarization of M1 macrophages after the overexpression of Col XV in mice. In addition, after overexpression of Col XV, the intracellular Ca²⁺ concentration was significantly increased. Using focal adhesion kinase (FAK) inhibitor PF573228, we found that PF-573228 inhibited the phosphorylation of FAK and reversed the upward trend of Col XV-induced protein expression levels of IRE1α, C/EBP-homologous protein (CHOP), and 78 kDa glucose-regulated protein (GRP78). After treatment with IRE1α inhibitor STF-083010, the results showed that the expression of adipocyte inflammation-related genes interleukin 6 (IL-6) and tumor necrosis factor α (TNFα) significantly were decreased. Our results demonstrate that Col XV induces ER-stress in adipocytes by activating the Integrinβ1/FAK pathway and disrupting the intracellular Ca²⁺ balance. At the same time, Col XV regulates the inflammation induced by ER stress in adipocytes by promoting IRE1α/XBP1 (X-Box binding protein 1) signaling. Our study provides new ideas for solving the problems of adipose tissue metabolism disorders caused by abnormal accumulation of ECM.

Keywords: Col XV; ERS; FAK; adipose tissue; inflammation.

Figure 1

Overexpression of Col XV promotes ERS in mouse adipocytes. (A) Heat map of genes up- or down-regulated in response to forced expression of Col XV in mouse adipocytes, and the most severely affected genes (n = 4). (B) GO analysis of the altered genes in (A) (n = 4) (left). Enrichment analysis of altered pathway in response to forced expression of Col XV (n = 4) (right). (C) Relative Col XV mRNA levels after knockdown and overexpression of Col XV. (D) Relative mRNA levels of Col XV in adipocytes pretreated with or without TM (1 μM) (n = 4). (E,F) Relative mRNA and protein expression levels of ERS marker genes in different groups in adipocyte pretreated with or without TM (1 μM) (n = 4). (G) The morphological changes of ER detected by ER red tracker (n = 4). Scale bar, 100 μm. Values are means ± SEM. ^# or * p < 0.05 or ** p < 0.01 compared with the control group.

Figure 2

Col XV promotes adipose inflammation. (A) Relative mRNA and protein expression levels of inflammation marker genes IL6, MCP1, TNFα in mice adipocytes in control group, pc-Col XV group and sh-Col XV group with or without TM (1μM) (n = 4). (B) Relative protein expression levels of IRE1α, TNFα, IL6, IL1β in different groups in adipocytes pretreated with or without TM (n = 4). (C) Relative protein expression levels of IRE1α, TNFα, IL6 in different groups in adipocytes treated with or without 4PBA (2ug/mL) (n = 4). (D) Relative Col XV mRNA level in iWAT of treatment group mice (n = 4). (E) Images of hematoxylin and eosin staining of white adipose tissue in different groups (n = 4). Scale bar, 100 μm. (F) Representative images of Masson’s trichrome staining in iWAT Arrowheads indicate collagen fibers (n = 4). Scale bar, 100 μm. (G) Images of immunohistochemical staining for F4/80 and TNFα of adipose tissue in different groups (n = 4). Scale bar, 200 μm. (H) Fluorescence staining of NLRP3 in adipocytes in different groups (n = 4). Scale bar, 100 μm. Values are means ± SEM. ^# or * p < 0.05 compared with the control group.

Figure 3

Col XV interacting with integrin β1 activates FAK. (A) The protein expression of integrin β1, FAK and p-FAK in different groups detected by Western blotting (n = 4). (B) Active FAK (FITC, green) in the cells is evaluated by immunofluorescence assay in different groups (n = 4). Scale bar, 200 μm. (C) Immunostaining of endogenous Col XV and endogenous integrin β1 in cells is performed for Col XV using an FITC-labeled secondary antibody, or integrin β1 using a Cy3-labeled secondary antibody (n = 4). (D) Confirmation of the interaction between Col XV and integrin β1 by co-IP (n = 4). (E,F) Relative protein and mRNA expressions of CHOP, IRE1α, GRP78 in different group (n = 4) after treatment with inhibitor of FAK PF573228. (G) Relative protein expression levels of p-FAK, FAK, IL6, TNFα (normalized to GAPDH) in each group (n = 4). Values are means ± SEM. ^# or * p < 0.05 and ^## or ** p < 0.01 compared with the control group.

Figure 4

Col XV disrupts intracellular Ca²⁺ homeostasis through IP3R1. (A) Analysis of [Ca²⁺] based on Fluo-3 AM mean fluorescence intensity after treatment assessed by fluorescence microscopy (n = 4). Scale bar, 200 μm. (B) FCM analysis of Cytosolic Ca²⁺. (C) Relative protein expressions of p-FAK, RYR, IP3R1 in adipocytes in the control group, pc-Col XV group and sh-Col XV groups (n = 4). (D) Relative protein expressions of IRE1α, CHOP, sXBP1, TNFα, IL6 in different groups incubated with 2APB (n = 4). (E) Relative mRNA levels of IP3R1, TNFα, XBP1 in adipocytes treated with PF-573228 (n = 4). (F) Relative protein levels of FAK, p-FAK and IP3R1 in adipocytes treated with or without PF-573228 (n = 4). Values are means ± SEM. ^# or * p < 0.05 and ** p < 0.01 compared with the control group.

Figure 5

IRE1α/XBP1 branch pathway participates in Col XV-induced inflammation. (A) Relative mRNA expression levels of PERK, ATF6 and IRE1α in different groups on adipocytes (n = 4). (B) Relative mRNA expression levels of PERK, ATF6 and IRE1α in different groups on iWAT (n = 4). (C) Representative images of IRE1α immunofluorescent staining in adipocytes (n = 4). Scale bar, 200 μm. (D) XBP1 mRNA splicing assay (RT-PCR) of indicated cells treated with PCR fragments corresponding to the uXBP1 or sXBP1 forms (n = 4). (E) Representative images of ER-tracker staining in different groups with or without inhibitor of IRE1α 4μ8c (Red) in adipocytes (n = 4). Scale bar, 200 μm. (F) Relative protein expression levels of IRE1α, sXBP1 and TNFα in different groups on adipocytes (n = 4). (G) Relative mRNA expression levels of IL6, TNFα, NLRP3 in adipocytes in different groups with or without STF083010 (n = 4). (H) Relative protein expression levels of IRE1α, sXBP1, TNFα, TRF2, p-NF-κB in adipocytes in different groups with or without inhibitor of IRE1α STF-083010 (n = 4). Values are means ± SEM. ^# or * p < 0.05 and ** p < 0.01 compared with the control group.

Figure 6

Col XV promotes adipose tissue M1 macrophage polarization. (A,B) 3T3-L1 cells are co-cultured with Raw264.7 cells by a trans-well (cell contact independent, soluble mediator driven) system. Relative mRNA and protein expression levels of IL6, TNFα, CD206, IL10 in RAW246.7 cells in different groups with or without the inhibitor of IRE1α STF083010 (n = 4). (C) Representative immunofluorescence method for iNOS in different groups (n = 4). Scale bar, 200 μm. (D,E) Relative mRNA expression levels of TNFα, IL6, MCP1 and protein expression levels of TNFα, IL6, IL1β in RAW246.7 cells incubated with the adipocyte control medium with or without the inhibitor of IRE1α STF-083010 (n = 4). (F,G) Relative mRNA expression levels of TNFα, IL6,F4/80,CD206 and relative protein expression levels of FAK, p-FAK, IL6, TNFα, IFNβ in mice iWAT in different groups (n = 4). Values are means ± SEM. ^# or * p < 0.05 compared with the control group.

Figure 7

Col XV increases ERS-mediated IFNβ secretin. (A) IFNβ expression level in medium supernatant in different groups with or without STF-083010 detected by ELISA (n = 4). (B) Relative protein expression levels of sXBP1, IFNβ in adipocytes in different groups with or without TM (n = 4). (C) Images of adipocytes INFβ stain by immunofluorescent staining in different groups (n = 4) Scale bar, 200 μm. (D) Representative images showing morphology of cells after labeling for F-actin (phalloidin-FITC, green) and nuclei (DAPI; blue) (n = 4). Scale bar, 100μm. (E) Relative protein expression levels of IRE1α, sXBP1, IFNβ in adipocytes in different groups with or without STF-083010 (n = 4). Values are means ± SEM. ^# or * p < 0.05 and ** p < 0.01 compared with the control group.