LPA1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferation

Abstract

There has been much recent interest in lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, in fibrotic diseases, but the mechanisms by which LPA-LPA1 signaling promotes pathological fibrosis remain to be fully elucidated. Using a mouse peritoneal fibrosis model, we demonstrate central roles for LPA and LPA1 in fibroblast proliferation. Genetic deletion or pharmacological antagonism of LPA1 protected mice from peritoneal fibrosis, blunting the increases in peritoneal collagen by 65.4 and 52.9%, respectively, compared to control animals and demonstrated that peritoneal fibroblast proliferation was highly LPA1 dependent. Activation of LPA1 on mesothelial cells induced these cells to express connective tissue growth factor (CTGF), driving fibroblast proliferation in a paracrine fashion. Activation of mesothelial cell LPA1 induced CTGF expression by inducing cytoskeleton reorganization in these cells, causing nuclear translocation of myocardin-related transcription factor (MRTF)-A and MRTF-B. Pharmacological inhibition of MRTF-induced transcription also diminished CTGF expression and fibrosis in the peritoneal fibrosis model, mitigating the increase in peritoneal collagen content by 57.9% compared to controls. LPA1-induced cytoskeleton reorganization therefore makes a previously unrecognized but critically important contribution to the profibrotic activities of LPA by driving MRTF-dependent CTGF expression, which, in turn, drives fibroblast proliferation.

Figure 1.

Genetic deletion or pharmacological inhibition of LPA₁ protects mice from CG-induced peritoneal fibrosis. A–D) Protection by genetic deletion of LPA₁. Data are expressed as means ± se. A) Masson's trichrome-stained peritoneal sections of WT (left) and LPA₁-KO mice (right). Representative sections are shown from untreated mice (d 0) and mice at d 21 of PBS or CG injections (×200). B) Peritoneal thickness of WT and LPA₁-KO mice following PBS or CG challenges (d 0, n=3 mice/genotype; d 21 PBS, n=8 mice/genotype; d 21 CG, n=8 mice/genotype). C) Biochemical analysis of CG-induced peritoneal fibrosis. Hydroxyproline content was measured in the peritoneum of WT and LPA₁-KO mice following CG or PBS challenges (d 21 PBS, n=6 mice/genotype; d 21 CG, n=6 mice/genotype). D) Peritoneal expression of COL1α₁ mRNA in WT and LPA₁-KO mice following CG or PBS challenges mice (d 21 PBS, n=5 mice/genotype; d 21 CG, n=5 mice/genotype). E–H) Protection by pharmacologic inhibition of LPA₁. Data are expressed as means ± se. E) Representative Masson's trichrome-stained peritoneal sections of vehicle-treated (left) and preventive AM095-treated mice (right, ×200). F) Peritoneal thickness following PBS or CG challenge (d 21 PBS, n=5 mice/treatment group; d 21 CG, n=5 mice/treatment group). V, vehicle treatment; P, preventive regimen of AM095; T, therapeutic regimen of AM095. G) Hydroxyproline content in the peritoneum following CG or PBS challenges (d 21 PBS, n=5 mice/treatment group; d 21 CG, n=5 mice/treatment group). H) Peritoneal expression of COLIα₁ mRNA following CG or PBS challenges (d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). Scale bars = 100 μm. *P < 0.01, **P < 0.05.

Figure 2.

CG-induced peritoneal αSMA⁺ myofibroblast accumulation is dependent on LPA₁. A) Peritoneal accumulation of αSMA⁺ myofibroblasts after CG challenges. Representative peritoneal sections of WT (left) and LPA₁-KO mice (right) stained with anti-αSMA antibody/peroxidase are shown from mice after 21 d of PBS or CG injections (×200). Scale bars = 100 μm. B) Numbers of αSMA⁺ cells in the submesothelial zone, expressed as mean number per HPF (d 21 PBS, n=5 mice/genotype; d 21 CG, n=5 mice/genotype). C) Peritoneal expression of αSMA mRNA in WT and LPA₁-KO mice following CG or PBS challenges, expressed as mean copies of αSMA mRNA relative to copies of GAPDH mRNA (d 21 PBS, n=5 mice/genotype; d 21 CG, n=5 mice/genotype). D) Peritoneal expression of αSMA mRNA following CG or PBS challenges in AM095-treated mice, expressed as mean copies of αSMA mRNA relative to copies of GAPDH mRNA (d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). V, vehicle; P, preventive AM095; T, therapeutic AM095. Data are expressed as means ± se. *P < 0.01.

Figure 3.

CG-induced peritoneal fibroblast accumulation and proliferation is dependent on LPA-LPA₁. A) Peritoneal accumulation of fibroblasts, proliferating cells, and proliferating fibroblasts after CG challenges. Representative peritoneal sections of vehicle-treated (top panel) and preventive AM095-treated COLI-GFP mice (bottom panel) stained with anti-GFP antibody (green) and anti-PCNA antibody (red) are shown (×400). Scale bars = 50 μm. B) Numbers of submesothelial GFP⁺ cells (fibroblasts), expressed as mean number per HPF (d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). C) Numbers of submesothelial GFP⁺PCNA⁺ cells (proliferating fibroblasts), expressed as mean number per HPF (d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). D) Percentages of submesothelial fibroblasts that are proliferating (GFP⁺PCNA⁺ cells/total GFP⁺ cells; d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). V, vehicle; P, preventive AM095. Data are expressed as means ± se. *P < 0.01.

Figure 4.

CG-induced CTGF expression is dependent on LPA₁ and is predominantly attributable to peritoneal mesothelial cells. A) Peritoneal expression of CTGF mRNA in WT and LPA₁-KO mice following CG or PBS challenges (d 21 PBS, n=5 mice/genotype; d 21 CG, n=5 mice/genotype). Data are expressed as mean copies of CTGF mRNA relative to copies of GAPDH mRNA. B) Peritoneal expression of CTGF protein in WT and LPA₁-KO mice following CG or PBS challenges (d 21 PBS, n=4 mice/genotype; d 21 CG, n=4 mice/genotype. Quantification was performed with ImageJ software; data are expressed as mean density of CTGF bands relative to GAPDH bands. C) Peritoneal expression of CTGF mRNA in vehicle and AM095-treated mice following CG or PBS challenges (d 21 PBS, n=6 mice/treatment group; d 21 CG, n=6 mice/treatment group). V, vehicle; P, preventive AM095; T, therapeutic AM095. Data are expressed as mean copies of CTGF mRNA relative to copies of GAPDH mRNA. D) Peritoneal location of CTGF protein in representative sections from WT and LPA₁-KO mice following PBS and CG challenges, stained with anti-CTGF antibody/peroxidase (×400). Arrowheads indicate CTGF⁺ mesothelial cells. Scale bars = 50 μm. Data are expressed as means ± se. *P < 0.01.

Figure 5.

Mesothelial cell-derived CTGF expression is dependent on LPA-LPA₁, and regulates fibroblast proliferation. A, B) LPA induces CTGF mRNA expression time dependently (A) and dose dependently (B) in PMCs (n=3 cell preparations/group). C, D) LPA induces CTGF protein expression time dependently (C) and dose dependently (D) in PMCs (n=2 cell preparations/group). E) LPA receptor expression of PMCs. Data are presented as copies of receptor mRNA relative to copies of β₂ microglobulin mRNA. F) LPA-induced CTGF mRNA expression was abrogated in LPA₁-KO PMCs (n=3 cell preparations/group). G) Identification of CTGF protein in CM from PMCs by Western blot. WT PMCs were transfected with control or CTGF siRNA, and LPA₁-KO PMCs with control siRNA. All PMCs were then stimulated with control medium or LPA (10 μM) for 24 h, and their CM was assayed for CTGF. H) NIH3T3 fibroblasts were transfected with CTGF siRNA, and then incubated with CM from the same groups of PMCs as in G. BrdU proliferation assays were performed after incubation with CM for 48 h (n=3 cell preparations/group), and expressed as mean OD value (OD_370–492). Data are expressed as means ± se. *P < 0.01.

Figure 6.

Mesothelial CTGF expression induced by LPA-LPA₁ signaling is independent of de novo protein synthesis, latent TGF-β activation, and Smad signaling. A) Effect of cycloheximide (CHX) on LPA-induced CTGF expression. PMCs were preincubated with CHX (20 μg/ml) or control medium for 2 h, and then stimulated with control medium or LPA (10 μM) for an additional 2 h (n=3 cell preparations/group). B) Effect of TGF-β neutralization on LPA-induced CTGF expression. PMCs were exposed to LPA (10 μM, for 2 h) or TGF-β₁ (1 ng/ml, for 4 h), with or without a pan-specific TGF-β neutralizing antibody (Ab, 10 μg/ml; n=3 cell preparations/group). C) Ability of LPA vs. TGF-β to induce Smad phosphorylation. PMCs were incubated with LPA (10 μM) or TGF-β₁ (5 ng/ml), and their lysates were assayed for phosphorylated Smad3 by Western blot. The experiment was performed in 2 independent series of PMC preparations. Data are expressed as mean ± sem copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA. *P < 0.01, **P < 0.05.

Figure 7.

Mesothelial CTGF expression induced by LPA-LPA₁ signaling is dependent on Gα_12/13 signaling, RhoA and ROCK activation, and actin polymerization. A, B) Validation of siRNA inhibition of Gα₁₂ and Gα₁₃ expression. PMCs were transfected with Gα₁₂, Gα₁₃, or control siRNA, and targeted Gα subunit inhibition was determined at mRNA (A) and protein (B) levels. mRNA data are expressed as copies of Gα₁₂ or Gα₁₃ mRNA relative to copies of β₂ microglobulin mRNA. C) Effects of Gα₁₂ and/or Gα₁₃ knockdown on LPA-induced CTGF expression, expressed as copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA. PMCs were transfected with control siRNA or Gα₁₂ and/or Gα₁₃ siRNA, and then stimulated with control medium or LPA (10 μM; n=3 cell preparations/group). D) Effect of pertussis toxin (PTX) on LPA-induced CTGF expression, expressed as copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA. PMCs were preincubated with PTX (100 ng/ml) or control medium for 18 h, and then stimulated for 2 h with control medium or LPA (10 μM). E) RhoA activation induced by LPA. Time course of active and total RhoA in PMCs following stimulation with LPA (10 μM). F) Dependence of LPA-induced RhoA activation on LPA₁ and Gα_12/13. WT PMCs were transfected with siRNAs targeting Gα₁₂ and Gα₁₃ or with control siRNA, and LPA₁-KO PMCs were transfected with control siRNA. Levels of active and total RhoA were then assayed 1 min after stimulation with control medium or LPA (10 μM; n=2 cell preparations/group). G) Dependence of LPA-induced CTGF expression on RhoA, ROCK, and actin polymerization. PMCs were preincubated with no inhibitor, or with C3 toxin (C3; 2.0 μg/ml for 10 h), Y27632 (Y; 5 μM for 30 min) or latrunculin B (LB; 1 μg/ml for 30 min). PMCs were then stimulated with LPA (10 μM) or control medium for an additional 2 h. Data are expressed as copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA (n=3 cell preparations/group). Data are expressed as means ± se. *P < 0.01.

Figure 8.

Y27632 (Y) and latrunculin B (LB) abrogate LPA-induced actin polymerization. A, B) Actin polymerization was visualized by immunocytochemical staining for phalloidin in PMCs that had been incubated in serum-free medium for 24 h and then stimulated for 30 min with control medium (A) or LPA (10 μM; B). C, D) PMCs were treated with Y27632 (Y; 5 μM; C) or latrunculin B (LB; 1 μg/ml; D) for 30 min prior to LPA stimulation. All images were captured using identical exposure settings. Scale bars = 50 μm.

Figure 9.

LPA promotes the nuclear translocation of MRTF-A and MRTF-B in a ROCK-dependent manner. A, C) Subcellular distributions of MRTF-A (A) and MRTF-B (C) were visualized by immunocytochemistry in PMCs that had been incubated in serum-free medium for 24 h and then stimulated with medium containing LPA (10 μM for 30 min) or control medium. Green represents MRTF-A or MRTF-B staining; blue represents DAPI staining. PMCs were additionally pretreated with Y27632 (Y; 5 μM) or control medium for 30 min before LPA stimulation. All images were captured using identical exposure settings. Scale bars = 100 μm. B, D) Quantification of the subcellular distribution of MRTF-A (B) and MRTF-B (D). Five random fields of view were counted per slide. Subcellular distributions were classified as nuclear (N; nuclear staining > cytoplasmic staining); equal (E; nuclear staining = cytoplasmic staining); or cytoplasmic (C; nuclear staining < cytoplasmic staining). Two independent series of PMCs were analyzed. All data are expressed as mean ± se distribution. Significant (P<0.01) comparisons for both MRTF-A and MRTF-B were percentages of cells with nuclear distributions, and percentages of cells with cytoplasmic distributions, in LPA alone vs. control, and LPA alone vs. LPA + Y27632.

Figure 10.

LPA-induced CTGF expression is dependent on MRTF-A, MRTF-B, and SRF signaling. A, B) Validation of siRNA inhibition of MRTF-A, MRTF-B, and SRF expression. PMCs were transfected with MRTF-A, MRTF-B, SRF, or control siRNA, and targeted transcription factor inhibition was determined at mRNA (A) and protein (B) levels. mRNA data are expressed as copies of MRTF-A, MRTF-B, or SRF mRNA relative to copies of β₂ microglobulin mRNA. C) Effects of MRTF-A, MRTF-B, or SRF knockdown on LPA-induced CTGF expression by PMCs. PMCs were transfected with control siRNA or siRNA targeting MRTF-A, MRTF-B, or SRF, and then stimulated with control medium or LPA (10 μM). mRNA data are expressed as copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA. (n=3 cell preparations/group). D) Effect of MRTF-SRF inhibition on LPA-induced CTGF expression. PMCs were preincubated with CCG-1423 (CCG; 10 μM) or control medium for 16 h, and then stimulated with LPA (10 μM) or control media for an additional 2 h. Data are expressed as copies of CTGF mRNA relative to copies of β₂ microglobulin mRNA (n=3 cell preparations/group). Data are expressed as means ± se. *P < 0.01.

Figure 11.

Pharmacological inhibition of the MRTF SRF-activating pathway attenuates peritoneal fibrosis. A) Representative Masson's trichrome-stained peritoneal sections of vehicle-treated (left panel) and CCG-1423-treated (3.0 mg/kg) mice (right panel) at d 21 of PBS or CG infections (×200). Scale bars = 100 μm. B) Peritoneal thickness following PBS or CG challenge (d 21 PBS, n=5 mice/treatment group; d 21 CG, n=5 mice/treatment group). Treatment groups are as indicated. C) Biochemical analysis of CG-induced peritoneal fibrosis. Peritoneal hydroxyproline content following CG or PBS challenges (d 21 PBS, n=5 mice/treatment group; d 21 CG, n=5 mice/treatment group). D–F) Peritoneal expression of CTGF, (D) αSMA (E), and zyxin and vinculin mRNA (F) following CG or PBS challenges (d 21 PBS, n=5 mice/treatment group; d 21 CG, n=5 mice/treatment group). Data are expressed as means ± se. *P < 0.01, **P < 0.05.

Figure 12.

Proposed profibrotic collaboration between mesothelial cells and fibroblasts in peritoneal fibrosis. LPA-LPA₁ signaling drives mesothelial cell CTGF expression through a Gα_12/13-RhoA-ROCK-MRTF-SRF pathway, and this mesothelial CTGF, in turn, drives fibroblast proliferation.