Silencing IQGAP1 alleviates hepatic fibrogenesis via blocking bone marrow mesenchymal stromal cell recruitment to fibrotic liver

Abstract

IQ motif-containing guanosine triphosphatase (GTPase)-activating protein 1 (IQGAP1) is a cytosolic scaffolding protein involved in cell migration. Our previous studies suggest sphingosine 1-phosphate (S1P) triggers bone marrow (BM) mesenchymal stromal cells (BMSCs) to damaged liver, thereby promoting liver fibrosis. However, the role of IQGAP1 in S1P-induced BMSC migration and liver fibrogenesis remains unclear. Chimeric mice of BM cell labeled by EGFP were used to build methionine-choline-deficient and high-fat (MCDHF)-diet-induced mouse liver fibrosis. IQGAP1 small interfering RNA (siRNA) was utilized to silence IQGAP1 in vivo. IQGAP1 expression is significantly elevated in MCDHF-diet-induced mouse fibrotic livers. Positive correlations are presented between IQGAP1 and fibrosis hallmarks expressions in human and mouse fibrotic livers. In vitro, depressing IQGAP1 expression blocks S1P-induced motility and cytoskeleton remodeling of BMSCs. S1P facilitates IQGAP1 aggregating to plasma membrane via S1P receptor 3 (S1PR₃) and Cdc42/Rac1. In addition, IQGAP1 binds to Cdc42/Rac1, regulating S1P-induced activation of Cdc42/Rac1 and mediating BMSC migration in concert. In vivo, silencing IQGAP1 reduces the recruitment of BMSCs to impaired liver and effectively alleviates liver fibrosis induced by MCDHF diet. Together, silencing IQGAP1 relieves liver fibrosis by blocking BMSC migration, providing an effective therapeutic strategy for liver fibrosis.

Keywords: Cdc42; F-actin; IQGAP1; Rac1; bone marrow mesenchymal stromal cells; liver fibrosis; migration; sphingosine 1-phosphate receptor 3.

Graphical abstract

Figure 1

The expression and localization of IQGAP1 during MCDHF-diet-induced liver injury Liver injury models were induced by methionine-choline-deficient and high-fat (MCDHF) diet. (A) Iqgap1 mRNA expression was studied in mouse livers with MCDHF diet feeding. (B) The protein level of IQGAP1 was detected in MCDHF-diet-fed mouse livers. (C and D) The correlations between mRNA levels of IQGAP1 and three liver fibrosis markers in mouse (C) or human (D) injured livers. (E) Immunofluorescence showed the localization of IQGAP1 in MCDHF-diet-induced fibrotic mouse livers. Scale bars: 25 μm. (F) Liver non-parenchymal cells (NPCs) (left) or hepatocytes (right) were isolated from mice fed with or without MCDHF diet. qRT-PCR was employed to detect IQGAP1 mRNA expression. (G) Representative images of immunofluorescence showed the expression of IQGAP1 and α-SMA in MCDHF-diet-induced liver fibrosis. DAPI was used for nuclear staining. Scale bars: 25 μm. Data are presented as the means ± SEM. ∗p < 0.05 versus control group (n = 6, per group).

Figure 2

IQGAP1 is involved in S1P-induced migration of BMSCs (A and B) The expression of IQGAP1 in BMSCs was detected by immunofluorescence (A) or western blot (B). HeLa cells was used as the positive control (B). Scale bars: 25 μm. (C) IQGAP1 was knocked down by small interfering RNA (siRNA) (40 nmol/L); the knockdown efficiency of IQGAP1 was studied by qRT-PCR (left) or western blot (right). (D) The migration ability of BMSCs was detected by Transwell chambers. (E) The representative images of BMSC migration. Scale bars: 100 μm. Arrow indicates migrating cell. (F) The representative images of microfilaments staining in BMSCs. Scale bars: 25 μm. BMSCs were stimulated with 10 nmol/L and 1 μmol/L S1P; the remodeling of microfilaments was examined by high-content analysis. (G and H) The fiber number (G) and fiber area (H) were analyzed by high-content analysis. Data are presented as the means ± SEM. All results were confirmed in three independent experiments at least. ∗p < 0.05 versus control group and #p < 0.05 versus S1P group (n = 3, per group).

Figure 3

IQGAP1 mediates S1P/S1PR₃-induced cell migration by aggregating to the cytoplasmic membrane (A) The localization of IQGAP1 in BMSCs treated with or without S1P. F-actin was also stained by FITC-labeled phalloidin. DAPI: nuclear staining. Scale bars: 10 μm. Arrowhead indicates activated-IQGAP1. (B) The number of IQGAP1-activated cells was quantified in S1P-treated BMSCs. (C) The number of IQGAP1-activated cells was quantified in S1P-treated BMSCs with preincubation of CAY-10444 (S1PR₃ inhibitor). (D) S1pr3 mRNA expression was knocked down in BMSCs detected by qRT-PCR. (E) The number of IQGAP1-activated cells was quantified in S1PR₃-silenced BMSCs with or without S1P. Data are presented as the means ± SEM. All results were confirmed in three independent experiments at least. ∗p < 0.05 versus control group and #p < 0.05 versus S1P group (n = 3, per group).

Figure 4

Cdc42 and Rac1 are involved in IQGAP1 plasma membrane aggregation BMSCs were stimulated with S1P. (A) Immunoprecipitation showed the binding between IQGAP1 and Cdc42/Rac1/actin, respectively. (B) Rho family (Cdc42, Rac1, and Rho) activation was studied in pull-down assay upon 60 min S1P treatment. (C) The migration ability of BMSCs after MLS-573151 (Cdc42 inhibitor) or NSC23766 (Rac1 inhibitor) treatment. (D) BMSCs were pre-treated with IQGAP1 siRNA, and S1P-induced Cdc42 or Rac1 activation was detected by pull-down assays. (E) NSC23766 or MLS-573151 was employed to block the function of Cdc42 or Rac1 in BMSCs, and BMSCs were treated with or without S1P. The number of IQGAP1-activated cells was quantified. Data are presented as the means ± SEM. All results were confirmed in three independent experiments at least. ∗p < 0.05 versus control group and #p < 0.05 versus S1P group (n = 3, per group).

Figure 5

Silencing IQGAP1 significantly reduced the migration of BMSCs to the injured liver (A and B) Mice injected with IQGAP1 siRNA were fed with MCDHF diet. The knockdown efficiency of liver and bone marrow was detected by qRT-PCR. (C) The mRNA expressions of Iqgap1 in kidney and heart were also detected. (D) The percentage of IQGAP1⁺ cells in NPCs or α-SMA⁺ cells was examined by flow cytometry. (E) The protein level of IQGAP1 was studied in the mouse liver described above. (F) Quantification of IQGAP1 protein expression levels. (G and H) Liver NPCs were isolated from mouse liver with or without IQGAP1 knockdown. Flow cytometric analysis was used to examine the number of CD146⁺, EGFP⁻CD146⁺, and EGFP⁺CD146⁺ cells in the fibrotic livers. The representative images of flow cytometric (G) and quantification of EGFP⁻CD146⁺ (H, left) and EGFP⁺CD146⁺ cells number (H, right) were shown. Data are presented as the means ± SEM. ∗p < 0.05 versus control group and #p < 0.05 versus MCDHF group with SCR siRNA (n = 5, per group).

Figure 6

Silencing of IQGAP1 significantly reduced myofibroblasts in fibrotic liver (A) The mRNA expression of Acta2 was studied in MCDHF-diet-fed mouse liver after injecting SCR or IQGAP1 siRNA. (B) The protein level of α-SMA in MCDHF-diet-fed mouse liver after injecting SCR or IQGAP1 siRNAs. (C) Flow cytometric analysis was used to examine the number of α-SMA⁺ cells in the fibrotic livers. The representative images of flow cytometric (left) and quantification of α-SMA⁺ cells number (right) were shown. (D–F) The contribution of BMSCs to myofibroblasts was studied by immunofluorescence. The representative images (D) and quantification (E and F) of α-SMA⁺ EGFP⁺ cells were shown. DAPI: nuclear staining. Scale bars: 50 μm. Data are presented as the means ± SEM. ∗p < 0.05 versus control group and #p < 0.05 versus MCDHF group with SCR siRNA (n = 5, per group).

Figure 7

Silencing of IQGAP1 significantly attenuated MCDHF-induced liver injury and fibrosis (A) Liver sections were subjected to Sirius Red staining. Scale bars: 50 μm. (B) The area of fibrotic liver was quantified. (C and D) mRNA levels of Col1a1 (C) and Col1a3 (D) were examined. (E and F) Serum levels of AST (E) and ALT (F) were measured in MCDHF-fed mice. (G) A schema diagram of IQGAP1-mediated, S1P-induced BMSC migration. Data are presented as the means ± SEM. ∗p < 0.05 versus control group and #p < 0.05 versus MCDHF group with SCR siRNA (n = 5, per group).