LPA1-induced cytoskeleton reorganization drives ﬁbrosis ... FASEB J 2013.pdf · -induced...

The FASEB Journal • Research Communication

LPA1-induced cytoskeleton reorganization drivesfibrosis through CTGF-dependentfibroblast proliferation

Norihiko Sakai,*,† Jerold Chun,§ Jeremy S. Duffield,�,¶,# Takashi Wada,**Andrew D. Luster,*,† and Andrew M. Tager*,†,‡,1

*Center for Immunology and Inflammatory Diseases, †Division of Rheumatology, Allergy, andImmunology, and ‡Pulmonary and Critical Care Unit, Massachusetts General Hospital, HarvardMedical School, Boston, Massachusetts, USA; §Department of Molecular Biology, Helen L. DorrisInstitute for Neurological and Psychiatric Disorders, Scripps Research Institute, La Jolla, California,USA; �Division of Nephrology, Department of Medicine, ¶Center for Lung Biology, and #Institute forStem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA; and**Division of Nephrology, Department of Laboratory Medicine, Institute of Medical, Pharmaceutical,and Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa, Japan

ABSTRACT There has been much recent interest inlysophosphatidic acid (LPA) signaling through one ofits receptors, LPA1, in fibrotic diseases, but the mech-anisms by which LPA-LPA1 signaling promotes patho-logical fibrosis remain to be fully elucidated. Using amouse peritoneal fibrosis model, we demonstrate cen-tral roles for LPA and LPA1 in fibroblast proliferation.Genetic deletion or pharmacological antagonism ofLPA1 protected mice from peritoneal fibrosis, bluntingthe increases in peritoneal collagen by 65.4 and 52.9%,respectively, compared to control animals and demon-strated that peritoneal fibroblast proliferation washighly LPA1 dependent. Activation of LPA1 on meso-thelial cells induced these cells to express connectivetissue growth factor (CTGF), driving fibroblast prolif-eration in a paracrine fashion. Activation of mesothelialcell LPA1 induced CTGF expression by inducing cyto-skeleton reorganization in these cells, causing nucleartranslocation of myocardin-related transcription factor(MRTF)-A and MRTF-B. Pharmacological inhibition ofMRTF-induced transcription also diminished CTGFexpression and fibrosis in the peritoneal fibrosismodel, mitigating the increase in peritoneal collagencontent by 57.9% compared to controls. LPA1-inducedcytoskeleton reorganization therefore makes a previ-

ously unrecognized but critically important contribu-tion to the profibrotic activities of LPA by drivingMRTF-dependent CTGF expression, which, in turn,drives fibroblast proliferation.—Sakai, N., Chun, J.,Duffield, J. S., Wada, T., Luster, A. D., Tager, A. M.LPA1-induced cytoskeleton reorganization drives fibro-sis through CTGF-dependent fibroblast proliferation.FASEB J. 27, 1830–1846 (2013). www.fasebj.org

Key Words: lysophosphatidic acid � Rho GTPases � myocardin-related transcription factor-A � myocardin-related transcrip-tion factor-B � serum response factor

Fibrosis characterizes many chronic diseases thatresult in end-stage organ failure, causing major mor-bidity and mortality. Fibrosis in many of these diseasesappears to result from aberrant or overexuberantwound-healing responses to injury (1), producing ex-cessive deposition of extracellular matrix. Expansion ofthe matrix-producing fibroblast pool is a critical com-ponent of profibrotic injury responses (2), but themolecular mediators driving this expansion in vivo havenot yet been fully identified. Better identification ofthese mediators will hopefully identify new therapeutictargets for fibrotic diseases, most of which are refractoryto currently available therapies.

Lysophosphatidic acid (LPA) is a bioactive lipid thatmediates diverse cellular responses through interactionswith specific G-protein-coupled receptors (GPCRs), ofwhich at least 5 have been definitively identified anddesignated LPA1–5 (3). We and others have recentlydemonstrated that LPA signaling specifically through

1 Correspondence: Center for Immunology and Inflamma-tory Diseases, Massachusetts General Hospital, 149 13th St.,Rm. 8301, Charlestown, MA 02129, USA; E-mail: [email protected]

doi: 10.1096/fj.12-219378

Abbreviations: �SMA, � smooth muscle actin; BAL, bron-choalveolar lavage; CG, chlorhexidine gluconate; CM, condi-tioned medium; COLI, type I collagen; COLI�1, �1 chain oftype I procollagen; CTGF, connective tissue growth factor;DMEM, Dulbecco’s modified Eagle’s medium; EGFP, enhancedgreen fluorescent protein; F-actin, filamentous actin; G-actin,globular actin; GFP, green fluorescent protein; GPCR, G-pro-tein-coupled receptor; HPF, high-power field; KO, knockout;LPA, lysophosphatidic acid; MRTF, myocardin-related tran-scription factor; PBS, phosphate-buffered saline; PCNA, pro-liferating cell nuclear antigen; PMC, peritoneal mesothelialcell; ROCK, Rho-associated coiled-coil-forming kinase; SRF,serum response factor; WT, wild-type

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LPA1 is required for the development of fibrosis inseveral organs (4–6), but the mechanisms throughwhich LPA-LPA1 signaling contributes to fibrosis re-main to be fully elucidated. Among its profibroticactivities, LPA-LPA1 signaling contributes to fibroblastaccumulation in pulmonary fibrosis by driving fibro-blast migration to sites of lung injury (4). Althoughfibroblast migration into the wound matrix is a centralstep in tissue responses to injury (7), recent evidencesuggests that the proliferation of resident fibroblastswithin injured tissues is central to the accumulation ofthese cells (8). We now demonstrate that fibroblastproliferation in vivo is dependent on LPA-LPA1 signal-ing, in a mouse model of fibrosis not previously shownto require LPA1, chlorhexidine gluconate (CG)-in-duced peritoneal fibrosis (9). Genetic deletion or phar-macological antagonism of LPA1 significantly attenu-ated fibroblast proliferation, as well as the developmentof fibrosis, in this model.

LPA itself can induce fibroblast proliferation, but thisdirect effect of LPA in vitro can be mediated by LPA1 orLPA2 (10). We therefore hypothesized that the specificrequirement for LPA1 that we observed for fibroblastproliferation in vivo was attributable to LPA signalingspecifically through LPA1 being required for the ex-pression of fibroblast mitogens after tissue injury otherthan LPA. Many of the activities induced by LPA, suchas cell migration and shape change, result, at least inpart, from its potent ability to regulate the actin cyto-skeleton through activation of the RhoA-Rho-associatedcoiled-coil-forming kinase (ROCK) cascade, convertingglobular actin (G-actin) monomers to filamentous actin(F-actin) polymers (11). The recent description of apathway linking actin cytoskeleton reorganization to geneexpression provides a potential mechanism throughwhich LPA-LPA1 signaling could induce fibroblastmitogen expression. In this pathway, polymerizationof G- into F-actin liberates myocardin-related tran-scription factor (MRTF)-A and MRTF-B, which thentranslocate to the nucleus and markedly augmentserum response factor (SRF)-induced gene transcrip-tion (12–13).

SRF is a MADS box transcription factor that inducesthe expression of genes by binding to CArG boxsequences [CC(A/T)6GG] in their promoters (14).The potent profibrotic mediator connective tissuegrowth factor (CTGF/CCN2) contains a CArG-like boxin its promoter, and SRF has been shown to induceCTGF expression in human endothelial cells (15).CTGF is highly expressed in multiple fibrotic patholo-gies (16) and induces fibroblast adhesion, extracellularmatrix production, and proliferation (17). Fibroblast-specific genetic deletion of CTGF was recently shown tomarkedly attenuate fibrosis and myofibroblast accumu-lation in the bleomycin model of scleroderma dermalfibrosis (18), a model that we have found to also behighly dependent on LPA-LPA1 signaling (5).

In this work, we investigated the hypotheses thatLPA-LPA1 signaling induces expansion of the fibroblastpool during the development of fibrosis in vivo; that

this expansion is driven by LPA-LPA1-induced CTGFexpression, which then drives fibroblast proliferation;and that this CTGF expression results from LPA-LPA1-induced actin polymerization, which liberates MRTF-Aand MRTF-B to translocate to the nucleus and augmentSRF-dependent transcription. This proposed link be-tween the profibrotic activities of LPA and CTGFconnects 2 important mediators that are each beingevaluated independently as therapeutic targets for mul-tiple fibrotic diseases.

The disease model we investigated in these studies,peritoneal fibrosis, is an important clinical problem inmultiple settings, including postsurgical peritoneal ad-hesions (19) and peritoneal dialysis (20), a life-sustain-ing therapy used by �100,000 patients with renalfailure worldwide, accounting for �10–15% of thedialysis population (21). Dialysis solutions that are hyper-osmotic, hyperglycemic, and/or acidic can cause repet-itive peritoneal injury (22), producing progressivefibrosis of the submesothelial region that normallyconsists of a thin layer of connective tissue, with a fewscattered fibroblasts, underlying a single-layered meso-thelial cell barrier (23). The development of peritonealfibrosis can markedly decrease dialysis ultrafiltrationcapacity and in some patients cause encapsulatingperitoneal sclerosis, which is associated with bowelobstruction and mortality rates as high as 38–56% (20,24–25). Elucidating the role of an LPA-LPA1-actin-MRTF-SRF-CTGF pathway will hopefully identify newtargets for therapies to treat and/or prevent peritonealfibrosis in all of the clinical settings in which it occurs.Characterizing this profibrotic pathway also will havethe potential to identify new therapeutic targets forfibrotic diseases affecting other organs in which LPA-LPA1 signaling and/or CTGF has been implicated.

MATERIALS AND METHODS

Reagents and cells

LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was purchasedfrom Avanti Polar Lipids (Alabaster, AL, USA) and diluted inDulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel,Switzerland) including 0.1% fatty acid-free bovine serumalbumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). AM095was the kind gift of Amira Pharmaceuticals (San Diego, CA,USA). Sodium pyruvate, NEAA mixture, and penicillin/streptomycin were obtained from Lonza. l-glutamine wasfrom Cellgro (Manassas, VA, USA). Y27632, pertussis toxin,cycloheximide, and DMSO were from Sigma-Aldrich. Latrun-culin B was from Invitrogen (Grand Island, NY, USA). CCG-1423 was from Cayman Chemical (Ann Arbor, MI, USA).Permeable C3 toxin was from Cytoskeleton (Denver, CO,USA). Recombinant TGF-�1, monoclonal anti-TGF-�1, -�2,and -�3 neutralizing antibody (1D11), and normal rabbit IgGwere from R&D Systems (Minneapolis, MN, USA). NIH3T3fibroblasts were purchased from American Type Culture Collec-tion (Manassas, VA, USA).

Mice

We purchased C57Bl/6 mice from the National CancerInstitute (NCI)-Frederick Mouse Repository (Frederick, MD,

1831AN LPA1-MRTF-CTGF PATHWAY TO FIBROSIS

USA). Experiments comparing LPA1-knockout (KO) andwild-type (WT) mice used offspring of mice heterozygous forthe LPA1 mutant allele, which were hybrids of the C57Bl/6and 129Sv/J genetic backgrounds (kindly provided by Dr.Jerold Chun, Scripps Research Institute, La Jolla, CA, USA;ref. 26). Experiments to identify fibroblasts used type Icollagen (COLI)–green fluorescent protein (GFP) trans-genic mice generated on the C57Bl/6 background (kindlyprovided by Dr. Jeremy Duffield, University of Washington,Seattle, WA, USA; ref. 27). All experiments used sex- andweight-matched mice at 6–10 wk of age maintained in specificpathogen-free environments and were performed in accor-dance with U.S. National Institutes of Health (NIH) guide-lines and protocols approved by the Massachusetts GeneralHospital Institutional Animal Care and Use Committee.

Peritoneal fibrosis model

Peritoneal fibrosis was induced by intraperitoneal injection of0.1% CG (Wako Pure Chemical Industries, Osaka, Japan)dissolved in 15% ethanol/phosphate buffered saline (PBS).CG was injected every other day over a period of 21 d.

Histology and peritoneal thickness measurement

A sample of peritoneal tissue from each mouse was fixed in10% buffered formalin (pH 7.2) and embedded in paraffin.We then stained 5-�m sections with Masson’s trichromeaccording to our standard protocol (4). Peritoneal thicknesswas measured from the junction of the parietal peritoneumwith the musculature of the abdominal wall to the serosalsurface of the parietal peritoneum, as described previously(9), on photomicrographs (�200) of Masson’s trichrome-stained sections at 5 randomly selected sites per high-powerfield (HPF) per section.

Hydroxyproline assay

Two pieces of peritoneal samples were taken by 6-mm punchbiopsy apparatus (Acuderm, Fort Lauderdale, FL, USA) fromeach mouse to assess peritoneal collagen, measured by itshydroxyproline content as determined by the standard pro-tocol of our laboratory (28). Assay results were expressed asmicrograms of hydroxyproline per piece.

RNA analyses

Total cellular RNA was isolated from primary cells usingRNeasy Mini Kits (Qiagen, Valencia, CA, USA). Total cellularRNA was isolated from peritoneal tissue by immersing thesurface of peritoneum in Trizol reagent (Invitrogen) for 20min and then extracting RNA according to the manufacturer’sinstructions. Quantitative real-time PCR analyses of RNAwere performed using Mastercycler realplex2 (Eppendorf,Hauppauge, NY, USA).

Immunohistochemical and immunocytochemical analyses

Myofibroblasts were identified in tissue samples using aspecific antibody against � smooth muscle actin (�SMA).Formalin-fixed, paraffin-embedded 5-�m sections were incu-bated with anti-mouse �SMA monoclonal antibody (E184;Abcam, Cambridge, MA, USA) followed by an Envision kit(Dako, Carpinteria, CA, USA). CTGF-expressing cells wereidentified using anti-mouse CTGF polyclonal antibodies(Santa Cruz Biotechnology, Santa Cruz, CA, USA). �SMA�

and CTGF� cells were visualized by incubating antibody-

stained sections with DAB (Dako). �-SMA� cells were thencounted in all fields of the submesothelial zone and ex-pressed as the mean � se number per HPF. To identifyproliferating fibroblasts, peritoneal sections from COLI-GFPmice were costained with anti-enhanced GFP (EGFP) mono-clonal antibody (Cell Signaling, Danvers, MA, USA) andanti-mouse proliferating cell nuclear antigen (PCNA) mono-clonal antibody (Abcam), using an M.O.M. kit (Vector Lab-oratories, Burlingame, CA, USA). Antibody-stained cells werevisualized using Fluorescein avidin (Vector Laboratories) forEGFP and Texas-red avidin (Vector Laboratories) for PCNA.EGFP and PCNA single-positive cells, and EGFP-PCNA dou-ble-positive cells, were then counted in all fields of thesubmesothelial zone and expressed as the mean � se numberper HPF.

Nuclear vs. cytoplasmic localization of MRTF-A and MRTF-Bwas assessed by immunocytochemical analyses performed onprimary mesothelial cells (PMCs), isolated as described be-low. PMCs were fixed in 4% paraformaldehyde in PBS,followed by the permeabilization with 0.2% Triton X-100 inPBS. Some PMCs were stimulated with LPA prior to fixation,and some cells were additionally treated with 5 �M Y27632 30min before LPA stimulation. Following permeabilization,PMCs were incubated with anti-mouse MRTF-A polyclonalantibodies (Santa Cruz Biotechnology) or anti-mouseMRTF-B polyclonal antibodies (Santa Cruz Biotechnology),followed by Alexa-Fluor 488-conjugated secondary antibodies(Invitrogen). Five random fields of view were counted, andsubcellular localization of MRTFs was scored as predomi-nantly nuclear, evenly distributed, or predominantly cytoplas-mic, as described previously (29).

Western blot analyses

Whole cellular lysates from primary cells were extracted withRIPA buffer (Thermo Scientific, Waltham, MA, USA) accord-ing to the manufacturer’s protocol. Samples of conditionedmedium (CM) were centrifuged to remove cellular debris,and concentrated (15-fold) using Amicon Ultra 10,000 (Mil-lipore, Billerica, MA, USA). Whole-tissue lysates were ex-tracted from peritoneal samples by immersing the surface ofperitoneal tissues in RIPA buffer (Thermo Scientific) for 20min. Cellular and tissue lysates, and concentrated CM, werethen separated by SDS-PAGE and transferred to polyvi-nylidene difluoride membranes (Invitrogen). After incuba-tion in blocking buffer containing 5% skim milk (Bio-Rad,Hercules, CA, USA), membranes were incubated overnight at4°C with rabbit anti-mouse CTGF polyclonal antibodies(Thermo Scientific), rabbit anti-mouse G�12 polyclonal anti-bodies (Santa Cruz Biotechnology), rabbit anti-mouse G�13polyclonal antibodies (Santa Cruz Biotechnology), rabbitanti-mouse MRTF-A polyclonal antibodies (Santa Cruz Bio-technology), rabbit anti-mouse MRTF-B polyclonal antibodies(Santa Cruz Biotechnology), rabbit anti-mouse SRF monoclo-nal antibody (Cell Signaling), rabbit anti-mouse Smad3monoclonal antibody (Cell Signaling), rabbit anti-mousephospho-Smad3 monoclonal antibody (Cell Signaling), orrabbit anti-mouse GAPDH monoclonal antibody (Cell Signal-ing). Following additional incubations of the membraneswith appropriate biotinylated secondary antibodies, proteinbands were detected with an enhanced chemiluminescentsubstrate (Thermo Scientific). Quantification was performedwith Image J software (NIH, Bethesda, MD, USA).

Inhibitor administration in vivo

The selective LPA1 antagonist AM095 was dissolved in sterilewater, and dosages of 30 mg/kg/mouse were administered by

1832 Vol. 27 May 2013 SAKAI ET AL.The FASEB Journal � www.fasebj.org

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oral gavage to COLI-GFP mice 2�/d on weekdays and 1�/d onweekends. Control COLI-GFP mice received equal volumes ofsterile water alone (vehicle) on the same schedule. AM095 orvehicle control was administered from the onset of CG chal-lenge in a preventive regimen, or beginning 7 d after the onsetof CG challenge in a therapeutic regimen.

The MRTF-SRF inhibitor CCG-1423 was dissolved invehicle (30% DMSO/15% ethanol/PBS), and a dose of 1.5or 3.0 mg/kg/mouse, or vehicle alone, was administered byintraperitoneal injection to C57Bl/6 mice. CCG-1423 dos-ages, or equal volumes of vehicle alone, were administered1�/d from the onset of CG challenge over a period of21 d.

Isolation of primary mouse PMCs

Primary PMCs were isolated from mice by enzymatic diges-tion of the inner surface of the peritoneum, as described

previously (30), with minor modifications. Briefly, parietalperitoneal flaps were removed and stretched on a sterileculture dish. RPMI 1640 (Lonza) containing 25 �g/mlLiberase (Roche, Basel, Switzerland) was placed on theperitoneal inner surface for 45 min at 37°C. After incuba-tion, the surface of the digested peritoneum was gentlyscraped to complete the release of partially attached me-sothelial cells. The cell pellet was collected by centrifuga-tion and resuspended in growth medium composed of 10%FBS-containing DMEM. To identify the outgrowing cells asmesothelial cells, immunocytochemistry with anti-vimentinantibody (VIM13.2; Sigma-Aldrich) and anti-cytokeratinantibody (PCK-26; Sigma-Aldrich) was performed. Morethan 98% of these cells were positive for vimentin andcytokeratin, consistent with their being mesothelial cells(31). In vitro experiments were performed on PMCs fromsecond to fifth passages.

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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 peritonealsections of WT (left) and LPA1-KO mice (right). Representative sections are shown from untreated mice (d 0) and miceat d 21 of PBS or CG injections (�200). B) Peritoneal thickness of WT and LPA1-KO mice following PBS or CG challenges(d 0, n3 mice/genotype; d 21 PBS, n8 mice/genotype; d 21 CG, n8 mice/genotype). C) Biochemical analysis ofCG-induced peritoneal fibrosis. Hydroxyproline content was measured in the peritoneum of WT and LPA1-KO micefollowing CG or PBS challenges (d 21 PBS, n6 mice/genotype; d 21 CG, n6 mice/genotype). D) Peritoneal expressionof COL1�1 mRNA in WT and LPA1-KO mice following CG or PBS challenges mice (d 21 PBS, n5 mice/genotype; d 21CG, n5 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-treatedmice (right, �200). F) Peritoneal thickness following PBS or CG challenge (d 21 PBS, n5 mice/treatment group; d 21CG, n5 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, n5 mice/treatment group; d21 CG, n5 mice/treatment group). H) Peritoneal expression of COLI�1 mRNA following CG or PBS challenges (d 21PBS, n6 mice/treatment group; d 21 CG, n6 mice/treatment group). Scale bars 100 �m. *P 0.01, **P 0.05.


RhoA activation assay

Quantification of RhoA activation in PMCs cultured in100-mm dishes was performed using a commercially availablekit (Cytoskeleton) according to the manufacturer’s instruc-tions.

siRNA transfections

In experiments using RNA interference, siRNAs targetingmouse MRTF-A, MRTF-B, SRF, and CTGF were On-TargetPlus Smart Pools, siRNAs targeting G�12 were an siGenomeSmart Pool (all from Thermo Scientific), and siRNAs target-ing G�13 were from Santa Cruz Biotechnology. On-TargetPlus nontargeting pool siRNAs were used as a nonspecificcontrol. PMCs or fibroblasts were transfected with siRNAs byLipofectamine 2000 (Invitrogen) according to the manufac-turer’s protocol and were incubated for 48 h prior to use inexperiments.

Fibroblast proliferation assay

Cultured PMCs were transfected with either CTGF-targetingor control siRNA and then stimulated with 10 �M LPA for 24 h.The CM was then aspirated, centrifuged to remove cellulardebris, and applied to serum-deprived NIH3T3 fibroblasts.Fibroblasts in these experiments were also treated with CTGF-targeting siRNA, to prevent any LPA remaining in the CMfrom stimulating fibroblast CTGF production. Fibroblast pro-liferation after incubation for 48 h with PMC-CM was deter-mined by BrdU assay (Roche), performed according to themanufacturer’s protocol.

Statistical analyses

Data are expressed as means � se. Unpaired t tests were usedfor comparisons between 2 groups, and analysis of variance

with post hoc Fisher’s test was used for comparisons between�2 groups. Values of P 0.05 were considered statisticallysignificant.

RESULTS

Genetic deletion of LPA1 protects mice fromCG-induced peritoneal fibrosis

LPA1-deficient (LPA1-KO) mice were dramatically pro-tected from CG-induced peritoneal fibrosis. Masson’strichrome staining demonstrated that collagen deposi-tion induced in WT mice by CG challenge was markedlyreduced in LPA1-KO mice (Fig. 1A). The degree ofprotection of LPA1-KO mice was quantified by measur-ing peritoneal thickness, hydroxyproline content, andmRNA levels of the �1 chain of type I procollagen(COLI�1). The peritoneal thickness of LPA1-KO micefollowing CG challenge was significantly less than thatof CG-challenged WT mice (55.3�4.7 vs. 116.0�6.0�m; Fig. 1B). In contrast, peritoneal thickness was notdifferent between LPA1-KO and WT mice challengedwith PBS (6.3�0.3 vs. 6.6�0.2 �m; Fig. 1B). Theincrease in peritoneal hydroxyproline content observedin CG-challenged WT mice was blunted by 65.4% inCG-challenged LPA1-KO mice (Fig. 1C), and the in-crease in peritoneal expression of COLI�1 mRNA wassignificantly reduced as well (Fig. 1D). These dataindicate that the LPA-LPA1 pathway importantly con-tributes to the development of peritoneal fibrosis.

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*Figure 2. CG-induced peritoneal �SMA� myofibroblast accumulation is depen-dent on LPA1. A) Peritoneal accumulation of �SMA� myofibroblasts after CGchallenges. Representative peritoneal sections of WT (left) and LPA1-KO mice(right) stained with anti-�SMA antibody/peroxidase are shown from mice after21 d of PBS or CG injections (�200). Scale bars 100 �m. B) Numbers of�SMA� cells in the submesothelial zone, expressed as mean number per HPF (d21 PBS, n5 mice/genotype; d 21 CG, n5 mice/genotype). C) Peritonealexpression of �SMA mRNA in WT and LPA1-KO mice following CG or PBSchallenges, expressed as mean copies of �SMA mRNA relative to copies ofGAPDH mRNA (d 21 PBS, n5 mice/genotype; d 21 CG, n5 mice/genotype).D) Peritoneal expression of �SMA mRNA following CG or PBS challenges inAM095-treated mice, expressed as mean copies of �SMA mRNA relative to copies

of GAPDH mRNA (d 21 PBS, n6 mice/treatment group; d 21 CG, n6 mice/treatment group). V, vehicle; P, preventiveAM095; T, therapeutic AM095. Data are expressed as means � se. *P 0.01.


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Pharmacological inhibition of LPA1 protects micefrom CG-induced peritoneal fibrosis

We next determined whether CG-induced peritonealfibrosis could be suppressed by administration of theLPA1-selective oral small molecule antagonist AM095(kind gift of Amira Pharmaceuticals; sodium{4=-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-acetate; ref. 5). AM095 was adminis-tered from CG challenge onset in a preventive regimento LPA1-sufficient mice. Since fibrosis is already estab-lished by d 7 in the CG model (32), we examined thepotential of LPA1 inhibition to treat peritoneal fibrosisby administering AM095 beginning 7 d after CG chal-lenge onset in a therapeutic regimen. To enable theanalyses of fibroblast accumulation and proliferationdescribed below, the LPA1-sufficient mice used in theseexperiments were COLI-GFP mice, in which fibroblastscan be identified by their transgenic expression ofEGFP driven by the fibroblast-specific collagen type I,

�1 promoter (27). Mice treated with AM095 in thepreventive regimen were dramatically protected fromperitoneal fibrosis, as indicated by Masson’s trichromestaining of peritoneal collagen (Fig. 1E), measurementsof peritoneal thickness (60.0�4.6 �m in preventiveA M 0 9 5 -treated mice vs. 108.5�4.8 �m in vehicle-treated CG-challenged mice; Fig. 1F), peritoneal hydroxyprolinecontent (Fig. 1G), and peritoneal COLI�1 mRNA ex-pression levels (Fig. 1H). The increase in peritonealhydroxyproline content observed in vehicle-treated CG-challenged mice was reduced by 52.9% in preventiveAM095-treated mice. Delayed administration of AM095in the therapeutic regimen also attenuated CG-inducedperitoneal thickness, hydroxyproline content, andCOLI�1 expression (Fig. 1F–H). Therapeutic AM095treatment mitigated the increase in peritoneal hydroxy-proline content of vehicle-treated CG-challenged miceby 41.2%. These data indicate an ongoing requirement

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Figure 3. CG-induced peritoneal fibroblast accumulation and proliferation is dependent on LPA-LPA1. A) Peritonealaccumulation of fibroblasts, proliferating cells, and proliferating fibroblasts after CG challenges. Representative peritonealsections of vehicle-treated (top panel) and preventive AM095-treated COLI-GFP mice (bottom panel) stained with anti-GFPantibody (green) and anti-PCNA antibody (red) are shown (�400). Scale bars 50 �m. B) Numbers of submesothelial GFP�

cells (fibroblasts), expressed as mean number per HPF (d 21 PBS, n6 mice/treatment group; d 21 CG, n6 mice/treatmentgroup). C) Numbers of submesothelial GFP�PCNA� cells (proliferating fibroblasts), expressed as mean number per HPF (d 21PBS, n6 mice/treatment group; d 21 CG, n6 mice/treatment group). D) Percentages of submesothelial fibroblasts that areproliferating (GFP�PCNA� cells/total GFP� cells; d 21 PBS, n6 mice/treatment group; d 21 CG, n6 mice/treatment group).V, vehicle; P, preventive AM095. Data are expressed as means � se. *P 0.01.


for the LPA-LPA1 pathway in the maintenance ofpathological peritoneal fibrosis, suggesting that target-ing this pathway may be an effective therapeutic strat-egy for peritoneal fibrosis.

CG-induced peritoneal �SMA� myofibroblastaccumulation is dependent on LPA1

Myofibroblasts are prominent in areas of increasedcollagen deposition in fibrotic tissues and are thoughtto be key effector cells producing pathological extra-cellular matrix in fibrotic diseases (33). We assessed theeffect of LPA-LPA1 signaling on the peritoneal accumu-lation of myofibroblasts, identified as �SMA-expressingcells by immunostaining. As demonstrated by the rep-resentative peritoneal sections in Fig. 2A, CG chal-lenges induced a robust, mainly submesothelial accu-mulation of �SMA� myofibroblasts. The increase inmyofibroblasts induced in WT mice by CG challengewas markedly attenuated in LPA1-KO mice (97.8�3.7vs. 38.2�3.4 cells/HPF, respectively; Fig. 2A, B). Theincrease in peritoneal expression of �SMA mRNAinduced by CG challenge was similarly reduced inLPA1-KO mice (Fig. 2C), as well as in mice treated withAM095 in either preventive or therapeutic regimens.These findings indicate that LPA signaling throughLPA1 is required for �SMA� myofibroblast accumula-tion during peritoneal fibrogenesis.

CG-induced peritoneal fibroblast accumulation andproliferation is dependent on LPA-LPA1

Although recruitment of bone marrow-derived progen-itors and transdifferentiation of other cells types mayplay important roles in increasing myofibroblast num-bers during the development of fibrosis, recent evi-dence suggests that the proliferation of fibroblastswithin injured tissues makes an essential contributionto myofibroblast accumulation (8). We compared fibro-blast proliferation induced by CG challenges in AM095-vs. vehicle-treated COLI-GFP mice. As demonstrated inthe representative sections in Fig. 3A, CG challengesinduced a marked peritoneal accumulation of GFP�

fibroblasts that was significantly reduced by LPA1 inhi-bition with AM095. The increased number of GFP�

fibroblasts present in CG-challenged, vehicle-treatedmice was significantly reduced in CG-challenged,AM095-treated mice (283.7�23.9 cells/HPF in CG-challenged, vehicle-treated mice vs. 73.5�13.4/HPF inCG-challenged, AM095-treated mice; Fig. 3B). To spe-cifically identify proliferating fibroblasts, we double-stained peritoneal sections with anti-PCNA antibodyand anti-GFP antibody. As demonstrated in the repre-sentative sections in Fig. 3A, fibroblast proliferationinduced by CG challenges was also dependent onLPA-LPA1 signaling. The number of proliferating (PCNA�)fibroblasts (GFP�) in the peritoneum after CG chal-

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Figure 4. CG-induced CTGF expression is dependent on LPA1and is predominantly attributable to peritoneal mesothelialcells. A) Peritoneal expression of CTGF mRNA in WT andLPA1-KO mice following CG or PBS challenges (d 21 PBS, n5mice/genotype; d 21 CG, n5 mice/genotype). Data are ex-pressed as mean copies of CTGF mRNA relative to copies ofGAPDH mRNA. B) Peritoneal expression of CTGF protein inWT and LPA1-KO mice following CG or PBS challenges (d 21PBS, n4 mice/genotype; d 21 CG, n4 mice/genotype. Quan-tification was performed with ImageJ software; data are ex-pressed as mean density of CTGF bands relative to GAPDHbands. C) Peritoneal expression of CTGF mRNA in vehicle andAM095-treated mice following CG or PBS challenges (d 21 PBS,n6 mice/treatment group; d 21 CG, n6 mice/treatmentgroup). 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 andLPA1-KO mice following PBS and CG challenges, stained with anti-CTGF antibody/peroxidase (�400). Arrowheadsindicate CTGF� mesothelial cells. Scale bars 50 �m. Data are expressed as means � se. *P 0.01.


www.fasebj.org

lenges was significantly higher in vehicle- vs. AM095-treated mice (158.3�17.2 vs. 23.0�4.6 cells/HPF; Fig.3C), as was the percentage of proliferating fibroblastsamong total fibroblasts (percentage of PCNA�GFP�

cells among total GFP� cells): 56.0 � 4.4 vs. 31.4 �2.6% in vehicle- vs. AM095-treated mice (Fig. 3D).These data suggest that reduced fibroblast proliferationin the absence of LPA1 signaling contributes to reducedperitoneal myofibroblast accumulation in LPA1-KOmice following CG challenges.

CG-induced CTGF expression requires LPA1, and isgenerated by peritoneal mesothelial cells

We hypothesized that LPA-LPA1 signaling drives fibro-blast proliferation after tissue injury in vivo at least inpart by driving the expression of important fibroblastmitogens other than LPA, such as CTGF. We focusedon CTGF for several reasons: CTGF stimulates fibro-blast proliferation (17, 34); fibroblast-specific deletionof CTGF markedly attenuates myofibroblast accumula-tion in the bleomycin model of dermal fibrosis (18);CTGF has been implicated in the pathogenesis of

peritoneal fibrosis in peritoneal dialysis patients (35);and CTGF has a CArG-like box in its promoter (15),which would allow it to be induced by an LPA-LPA1-actin-MRTF-SRF pathway. CG challenges markedly in-duced peritoneal CTGF mRNA (Fig. 4A) and protein(Fig. 4B) expression, both of which were significantlyattenuated in LPA1-KO mice. Peritoneal CTGF mRNAexpression was also significantly suppressed by theLPA1 antagonist AM095 administered in either preven-tive or therapeutic regimens (Fig. 4C). These dataindicate that LPA-LPA1 signaling is required for theinduction of CTGF expression during the developmentof peritoneal fibrosis. To determine the cellular sourcesof CTGF in this model, we identified CTGF� cells byimmunostaining. Robust CTGF staining was detected inthe PMCs of CG-challenged WT mice, with reducedstaining being detected in the PMCs of LPA1-KO mice(Fig. 4D). Although some CTGF staining was alsodetected in cells in the expanded peritoneal intersti-tium of CG-challenged mice, the robust expression ofCTGF by PMCs is consistent with accumulating evi-dence that these cells are a critically important source

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Figure 5. Mesothelial cell-derived CTGFexpression is dependent on LPA-LPA1, andregulates fibroblast proliferation. A, B) LPAinduces CTGF mRNA expression time de-pendently (A) and dose dependently (B) inPMCs (n3 cell preparations/group). C,D) LPA induces CTGF protein expressiontime dependently (C) and dose depend-ently (D) in PMCs (n2 cell preparations/group). E) LPA receptor expression ofPMCs. Data are presented as copies ofreceptor mRNA relative to copies of �2microglobulin mRNA. F) LPA-induced

CTGF mRNA expression was abrogated in LPA1-KO PMCs (n3 cell preparations/group). G) Identification of CTGFprotein in CM from PMCs by Western blot. WT PMCs were transfected with control or CTGF siRNA, and LPA1-KO PMCswith control siRNA. All PMCs were then stimulated with control medium or LPA (10 �M) for 24 h, and their CM was assayedfor CTGF. H) NIH3T3 fibroblasts were transfected with CTGF siRNA, and then incubated with CM from the same groupsof PMCs as in G. BrdU proliferation assays were performed after incubation with CM for 48 h (n3 cell preparations/group), and expressed as mean OD value (OD370–492). Data are expressed as means � se. *P 0.01.


of profibrotic molecules, including cytokines, growthfactors, and matrix proteins, in the pathogenesis ofperitoneal fibrosis (36).

LPA-LPA1 signaling induces PMC CTGF expression,which induces fibroblast proliferation

To investigate the ability of LPA-LPA1 signaling todirectly induce PMC CTGF expression, we isolatedprimary mouse PMCs for in vitro studies. LPA inducedrobust CTGF mRNA expression in primary PMCs iso-lated from WT mice (WT-PMCs) in a time- and dose-dependent manner (Fig. 5A, B). Maximal CTGF mRNAinduction was observed 2 h after stimulation with 10�M LPA. LPA similarly induced CTGF protein produc-tion by WT-PMCs, also in a time- and dose-dependentmanner (Fig. 5C, D). To determine which LPA recep-tors are responsible for LPA-induced CTGF expressionby WT-PMCs, we first determined the LPA receptorexpression profile of these cells. Of the 5 definitivelyidentified LPA receptors, LPA1 was the most highlyexpressed by WT-PMCs (LPA1�LPA2�LPA3�LPA4�LPA5; Fig. 5E). Primary mouse PMCs isolated fromLPA1-KO mice (LPA1-KO PMCs) did not express LPA1as expected, and their LPA1 deficiency did not causecompensatory changes in their expression of other LPAreceptors (Fig. 5E). LPA-induced CTGF mRNA expres-sion was almost completely abrogated in LPA1-KOPMCs (Fig. 5F), indicating that signaling through LPA1is predominantly responsible for CTGF induction byLPA.

To investigate the potential role of CTGF as a down-stream mediator of LPA’s ability to drive peritonealfibroblast proliferation, we investigated the ability ofmedium conditioned by LPA-stimulated PMCs to in-duce fibroblast proliferation, and whether any of theproliferation induced was attributable to CTGF pro-

duced by PMCs in response to LPA-LPA1 signaling.Medium conditioned by LPA-stimulated PMCs con-tained increased CTGF (Fig. 5G) and stimulated in-creased fibroblast proliferation (Fig. 5H), comparedwith medium conditioned by unstimulated PMCs.CTGF content and fibroblast proliferative activity ofmedium conditioned by LPA-stimulated PMCs weresimultaneously reduced when PMCs were transfectedwith siRNAs targeting CTGF, or when PMCs wereisolated from LPA1-KO mice (Fig. 5G, H). To preventany LPA remaining in the CM from directly stimulatingCTGF expression by the responding fibroblasts, theresponding fibroblasts in these experiments were alsotransfected with siRNA targeting CTGF. Taken to-gether, these data support the hypothesis that LPA-LPA1 signaling promotes fibroblast proliferation dur-ing the development of peritoneal fibrosis by inducingCTGF expression by PMCs.

LPA-induced CTGF expression is independent of denovo protein synthesis and TGF-� activation

CTGF expression is highly induced by TGF-� (37), andLPA has been demonstrated to induce activation oflatent TGF-� by lung epithelial cells (38) and smoothmuscle cells (39). To investigate whether LPA-inducedCTGF expression required de novo production ofTGF-�, or any other proteins in an autocrine fashion,we investigated whether this CTGF expression wassensitive to inhibition of protein synthesis with cyclo-heximide. As shown in Fig. 6A, cycloheximide treat-ment did not block LPA-induced CTGF mRNA expres-sion by PMCs, actually resulting in CTGF “superinduction,”i.e., augmented mRNA induction following agoniststimulation in the presence of translational blockers,such as cycloheximide (40). LPA-induced CTGF expres-sion in PMCs was therefore independent of new TGF-� or

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Figure 6. Mesothelial CTGF expression induced by LPA-LPA1 signaling is independent of de novo protein synthesis, latent TGF-�activation, and Smad signaling. A) Effect of cycloheximide (CHX) on LPA-induced CTGF expression. PMCs were preincubatedwith CHX (20 �g/ml) or control medium for 2 h, and then stimulated with control medium or LPA (10 �M) for an additional2 h (n3 cell preparations/group). B) Effect of TGF-� neutralization on LPA-induced CTGF expression. PMCs were exposedto LPA (10 �M, for 2 h) or TGF-�1 (1 ng/ml, for 4 h), with or without a pan-specific TGF-� neutralizing antibody (Ab, 10 �g/ml;n3 cell preparations/group). C) Ability of LPA vs. TGF-� to induce Smad phosphorylation. PMCs were incubated with LPA(10 �M) or TGF-�1 (5 ng/ml), and their lysates were assayed for phosphorylated Smad3 by Western blot. The experiment wasperformed in 2 independent series of PMC preparations. Data are expressed as mean � sem copies of CTGF mRNA relative tocopies of �2 microglobulin mRNA. *P 0.01, **P 0.05.


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other protein production. Since TGF-� activity in vivo isprimarily regulated by the post-translational conversion oflatent TGF-� complexes to active TGF-� (41), we investi-gated whether LPA induced CTGF expression by activat-ing latent TGF-�. Treatment with pan-specific TGF-�-neutralizing antibody had no effect on LPA-inducedCTGF expression (Fig. 6B), and LPA stimulation did notinduce phosphorylation of Smad3 (Fig. 6C). Taken to-gether, these results suggest that PMC CTGF expressioninduced by the LPA-LPA1 pathway is independent ofTGF-� production, activation, or canonical Smad signal-ing.

LPA-induced CTGF expression is dependent onG�12/13, RhoA, ROCK, and actin polymerization

LPA receptors couple to different classes of G proteins,including those containing G�12/13, G�i/o, or G�q �

subunits, to mediate the diverse activities of LPA (11).Transfection of PMCs with siRNAs targeting eitherG�12 or G�13 demonstrated the involvement of G�12/13-containing G proteins in LPA-induced CTGF expres-sion. The extent of siRNA-induced suppression of G�12or G�13 expression in PMCs is shown Fig. 7A, B, and theability of these siRNAs to significantly suppress LPA-induced PMC CTGF expression is shown in Fig. 7C. Incontrast, pretreatment of PMCs with pertussis toxin,which inhibits G�i/o–containing G proteins, had nosignificant effect on LPA-induced CTGF expression(Fig. 7D).

The effects of LPA receptor G-protein-coupled sig-naling on the actin cytoskeleton are mediated by Rhofamily of GTPases, including RhoA and Rac (11).Whereas G�i/o G proteins typically mediate LPA-in-duced Rac activation, G�12/13 G proteins are typicallyresponsible for LPA-induced activation of RhoA, and

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Figure 7. Mesothelial CTGF expression induced by LPA-LPA1 signaling is dependent on G�12/13 signaling, RhoA and ROCKactivation, and actin polymerization. A, B) Validation of siRNA inhibition of G�12 and G�13 expression. PMCs were transfectedwith G�12, G�13, or control siRNA, and targeted G� subunit inhibition was determined at mRNA (A) and protein (B) levels.mRNA data are expressed as copies of G�12 or G�13 mRNA relative to copies of �2 microglobulin mRNA. C) Effects of G�12and/or G�13 knockdown on LPA-induced CTGF expression, expressed as copies of CTGF mRNA relative to copies of �2microglobulin mRNA. PMCs were transfected with control siRNA or G�12 and/or G�13 siRNA, and then stimulated with controlmedium or LPA (10 �M; n3 cell preparations/group). D) Effect of pertussis toxin (PTX) on LPA-induced CTGF expression,expressed as copies of CTGF mRNA relative to copies of �2 microglobulin mRNA. PMCs were preincubated with PTX (100ng/ml) or control medium for 18 h, and then stimulated for 2 h with control medium or LPA (10 �M). E) RhoA activationinduced by LPA. Time course of active and total RhoA in PMCs following stimulation with LPA (10 �M). F) Dependence ofLPA-induced RhoA activation on LPA1 and G�12/13. WT PMCs were transfected with siRNAs targeting G�12 and G�13 or withcontrol siRNA, and LPA1-KO PMCs were transfected with control siRNA. Levels of active and total RhoA were then assayed 1min after stimulation with control medium or LPA (10 �M; n2 cell preparations/group). G) Dependence of LPA-inducedCTGF expression on RhoA, ROCK, and actin polymerization. PMCs were preincubated with no inhibitor, or with C3 toxin (C3;2.0 �g/ml for 10 h), Y27632 (Y; 5 �M for 30 min) or latrunculin B (LB; 1 �g/ml for 30 min). PMCs were then stimulated withLPA (10 �M) or control medium for an additional 2 h. Data are expressed as copies of CTGF mRNA relative to copies of �2microglobulin mRNA (n3 cell preparations/group). Data are expressed as means � se. *P 0.01.


we consequently hypothesized that LPA-induced CTGFexpression would depend on RhoA. We found that LPAstimulation of WT-PMCs activated RhoA, with the great-est activation at early times (1 min) poststimulation(Fig. 7E). LPA-induced RhoA activation was suppressedin LPA1-KO PMCs, and in WT-PMCs transfected withG�12 and G�13 siRNAs (Fig. 7F), indicating that LPA-induced RhoA activation in PMCs is mediated by LPA1and G�12/13 signaling. LPA-induced CTGF expressionwas significantly attenuated by treatment of PMCs withthe RhoA inhibitor C3 toxin (Fig. 7G), which reducedbasal levels of CTGF mRNA in PMCs as well, indicatingthat LPA’s ability to activate RhoA is required for itsability to induce CTGF expression.

RhoA signaling induces focal adhesion formation,actin polymerization, and stress fiber formation byactivating ROCKs. LPA-induced CTGF expression wassignificantly attenuated by treatment of PMCs with thenonselective ROCK inhibitor Y-27632, as well as by theactin polymerization inhibitor latrunculin B (Fig. 7G).Both Y-27632 and latrunculin B reduced basal levels ofCTGF mRNA in PMCs as well. Both of these inhibitorsabrogated actin polymerization induced by LPA. Usingphalloidin to visualize polymeric F-actin, LPA stimula-tion induced F-actin formation in PMCs as expected(Fig. 8A, B), and this actin polymerization was com-pletely inhibited by pretreatment with Y27632 or latrun-culin B (Fig. 8C, D). Taken together, these data indicatethat LPA-LPA1-induced CTGF expression is mediatedby a signaling pathway involving G�12/13, RhoA, ROCK,and actin polymerization.

LPA promotes the nuclear translocation of MRTF-Aand MRTF-B

MRTF-A and MRTF-B have recently been identified asnovel links between actin dynamics and gene expres-sion (13). The MRTFs form stable complexes with

monomeric G-actin, resulting in their sequestration inthe cytoplasm. Polymerization of G-actin into the F-ac-tin filaments liberates MRTFs, which then translocateto the nucleus. In the nucleus, the MRTFs act ascofactors for SRF, augmenting the activity of this nu-clear transcription factor (13). We therefore hypothe-sized that the LPA-LPA1-G�12/13-RhoA-ROCK-actin sig-naling pathway induces gene expression by promotingthe nuclear translocation of MRTF-A and MRTF-B. LPAstimulation of PMCs significantly decreased the per-centage of cells with predominantly cytoplasmic local-ization of MRTF-A from 28.5 to 6.4%, and significantlyincreased the percentage of cells with predominantlynuclear MRTF-A from 44.3 to 86.6% (Fig. 9A, B).Similarly, LPA stimulation of PMCs significantly de-creased the percentage of cells with predominantlycytoplasmic MRTF-B from 60.4 to 33.5%, and signifi-cantly increased the number of cells with predomi-nantly nuclear MRTF-B from 18.0 to 41.2% (Fig. 9C, D).Consistent with LPA-induced MRTF nuclear transloca-tion being mediated through the LPA1-G�12/13-RhoA-ROCK-actin pathway, PMC pretreatment with Y-27632markedly blocked LPA-induced MRTF nuclear translo-cation, reducing the percentages of cells with predom-inantly nuclear MRTF-A and MRTF-B to below controllevels (Fig. 9).

LPA-induced CTGF expression in PMCs is dependenton MRTF-A, MRTF-B, and SRF

Transfection of PMCs with siRNAs targeting MRTF-A,MRTF-B, or SRF demonstrated the involvement ofthese transcription factors in LPA-induced CTGFexpression. The extent of siRNA-induced suppres-sion of MRTF-A, MRTF-B, and SRF in PMCs is shownat the mRNA and protein levels in Fig. 10A, B,respectively, and the ability of these siRNAs to signif-icantly suppress LPA-induced PMC CTGF expression

Control LPA

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Figure 8. Y27632 (Y) and latrunculin B (LB)abrogate LPA-induced actin polymerization. A, B)Actin polymerization was visualized by immunocy-tochemical staining for phalloidin in PMCs thathad been incubated in serum-free medium for 24 hand then stimulated for 30 min with controlmedium (A) or LPA (10 �M; B). C, D) PMCs weretreated with Y27632 (Y; 5 �M; C) or latrunculin B(LB; 1 �g/ml; D) for 30 min prior to LPAstimulation. All images were captured using iden-tical exposure settings. Scale bars 50 �m.


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is shown in Fig. 10C. To confirm that LPA-inducedCTGF expression requires MRTF-SRF signaling, wetreated PMCs with CCG-1423 (N-[2-[4(4-chlorophenyl)-amino]-1-methyl-2-oxoethoxy]-3,5-bis-(trifluoromethyl)-benzamide), a small molecule inhibitor of the MRTF-activating pathway of SRF-dependent transcription(42). Treatment of PMCs with CCG-1423 abrogatedLPA-induced CTGF expression, and suppressed basallevels of CTGF mRNA as well (Fig. 10D). Takentogether, these results suggest that the MRTF-SRFcircuit of transcriptional regulation is also part of thesignaling pathway through which LPA induces CTGFexpression in PMCs.

Pharmacological inhibition of the MRTF-activatingpathway of SRF-dependent transcription attenuatesperitoneal fibrosis

To determine the relevance of the MRTF-SRF-activat-ing pathway to peritoneal fibrosis in vivo, we evaluatedCCG-1423 in the CG-induced peritoneal fibrosis model.CCG-1423 treatment significantly suppressed CG-in-duced increases in peritoneal collagen staining, thick-ness, and hydroxyproline content observed in vehicle-treated control mice (Fig. 11A–C). The increases inperitoneal hydroxyproline content observed in vehicle-treated CG-challenged mice were reduced by 52.6 and

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Figure 9. LPA promotes the nuclear translocation of MRTF-A and MRTF-B in a ROCK-dependent manner. A, C) Subcellulardistributions of MRTF-A (A) and MRTF-B (C) were visualized by immunocytochemistry in PMCs that had been incubated inserum-free medium for 24 h and then stimulated with medium containing LPA (10 �M for 30 min) or control medium. Greenrepresents MRTF-A or MRTF-B staining; blue represents DAPI staining. PMCs were additionally pretreated with Y27632 (Y; 5�M) or control medium for 30 min before LPA stimulation. All images were captured using identical exposure settings. Scalebars 100 �m. B, D) Quantification of the subcellular distribution of MRTF-A (B) and MRTF-B (D). Five random fields of viewwere 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 independentseries of PMCs were analyzed. All data are expressed as mean � se distribution. Significant (P0.01) comparisons for bothMRTF-A and MRTF-B were percentages of cells with nuclear distributions, and percentages of cells with cytoplasmicdistributions, in LPA alone vs. control, and LPA alone vs. LPA � Y27632.


57.9% in mice treated with CCG-1423 at 1.5 and 3.0mg/kg dosages, respectively. CG-induced increases inperitoneal expression of CTGF and �SMA were alsosignificantly reduced in CCG-1423-treated mice (Fig.11D, E). We also demonstrated inhibition of peritonealexpression of other known SRF target genes, includingzyxin and vinculin, in CCG-1423-treated mice, provid-ing evidence that CCG-1423 inhibits SRF-induced tran-scription in these in vivo experiments (Fig. 11F). Takentogether, these data implicate the MRTF-SRF-activatingpathway in the pathogenesis of peritoneal fibrosis invivo.

DISCUSSION

We found that LPA-LPA1 signaling is required for thedevelopment of peritoneal fibrosis. Genetic deletion orpharmacological antagonism of LPA1 protected micefrom fibrosis produced by CG peritoneal injury. Dis-

rupting the LPA-LPA1 pathway dramatically reducedperitoneal CTGF expression, fibroblast proliferation,and peritoneal myofibroblast accumulation. LPA en-gagement of PMC LPA1 induced CTGF expression,which accounted for the majority of the fibroblastproliferative activity produced by LPA-treated PMCs.LPA engagement of PMC LPA1 induced CTGF expres-sion through G�12/13 signaling, RhoA and ROCK acti-vation, actin polymerization, MRTF-A and MRTF-B nu-clear translocation, and SRF-induced transcription.Pharmacological inhibition of MRTF-SRF-induced tran-scription reduced CTGF expression and peritoneal fibro-sis in the CG model. We believe these data place LPA-LPA1 signaling at the center of a profibrotic collaborationbetween mesothelial cells and fibroblasts in the develop-ment of peritoneal fibrosis (Fig. 12), in which LPA drivesmesothelial cell CTGF expression, and this mesothelialCTGF in turn drives fibroblast proliferation.

We had previously found evidence that LPA1 ex-pressed by fibroblasts contributes to the recruitment of

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Figure 10. LPA-induced CTGF expression is dependent on MRTF-A, MRTF-B, and SRF signaling. A, B) Validation of siRNAinhibition of MRTF-A, MRTF-B, and SRF expression. PMCs were transfected with MRTF-A, MRTF-B, SRF, or control siRNA, andtargeted transcription factor inhibition was determined at mRNA (A) and protein (B) levels. mRNA data are expressed as copiesof MRTF-A, MRTF-B, or SRF mRNA relative to copies of �2 microglobulin mRNA. C) Effects of MRTF-A, MRTF-B, or SRFknockdown 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 �M). mRNA data are expressed as copies of CTGFmRNA relative to copies of �2 microglobulin mRNA. (n3 cell preparations/group). D) Effect of MRTF-SRF inhibition onLPA-induced CTGF expression. PMCs were preincubated with CCG-1423 (CCG; 10 �M) or control medium for 16 h, and thenstimulated with LPA (10 �M) or control media for an additional 2 h. Data are expressed as copies of CTGF mRNA relative tocopies of �2 microglobulin mRNA (n3 cell preparations/group). Data are expressed as means � se. *P 0.01.


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these cells during the development of pulmonary fibro-sis (4). In contrast, we found that fibroblast LPA1expression is not required for the proliferation of thesecells in response to lung injury. We found that most ofthe fibroblast migration induced by bronchoalveolarlavage (BAL) from mice in the bleomycin model ofpulmonary fibrosis was lost when the responding fibro-blasts were LPA1 deficient. In contrast, we found nosignificant differences between the proliferative re-sponses of WT and LPA1-deficient fibroblasts to BALfrom bleomycin-challenged mice (4). Our current datain the CG model of peritoneal fibrosis suggests anoncell autonomous requirement of LPA1 for fibro-blast proliferation in vivo: LPA1 expression by PMCs isrequired for their expression of CTGF, which inducesfibroblast proliferation in a paracrine fashion. Thisproposed mesothelial cell-fibroblast profibrotic cross-talk is consistent with accumulating evidence that para-crine interactions between multiple cell types are cen-tral to the development of fibrosis (1).

There have been prior indications that LPA may bean important regulator of CTGF expression. Similar toour findings in peritoneal fibrosis, CTGF expressionhas previously been demonstrated to be LPA depen-

dent in a mouse model of pulmonary fibrosis inducedby radiation (43), and similar to our findings withPMCs, LPA has been shown to induce CTGF expressionin mouse skeletal muscle cells (44). The molecularpathways through which LPA induces CTGF may be celltype dependent, however. LPA-induced skeletal muscleCTGF expression required TGF-� receptor signalingand Smad 2/3 expression (44), whereas we found thattreatment of PMCs with a pan-TGF-� neutralizing anti-body had no effect on their CTGF expression inducedby LPA. As we have described, this TGF-�-independentpathway through which LPA induces CTGF in PMCssequentially involves LPA1 and G�12/13 signaling, RhoAand ROCK activation, actin polymerization, MRTF-Aand MRTF-B nuclear translocation, and SRF-inducedtranscription. Although the ability of LPA and RhoA toinduce SRF-dependent transcriptional activation hasalso been reported (45), to our knowledge the molec-ular connections between LPA-induced RhoA-medi-ated cytoskeletal rearrangements and SRF activationhave not been previously described. Similarly, the im-portance of LPA-induced SRF activation to CTGF-induced fibroblast proliferation and the developmentof pathological fibrosis have not previously been appre-

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Figure 11. Pharmacological inhibition of the MRTF SRF-activating pathway attenuates peritoneal fibrosis. A) RepresentativeMasson’s trichrome-stained peritoneal sections of vehicle-treated (left panel) and CCG-1423-treated (3.0 mg/kg) mice (rightpanel) at d 21 of PBS or CG infections (�200). Scale bars 100 �m. B) Peritoneal thickness following PBS or CG challenge(d 21 PBS, n5 mice/treatment group; d 21 CG, n5 mice/treatment group). Treatment groups are as indicated. C)Biochemical analysis of CG-induced peritoneal fibrosis. Peritoneal hydroxyproline content following CG or PBS challenges (d21 PBS, n5 mice/treatment group; d 21 CG, n5 mice/treatment group). D–F) Peritoneal expression of CTGF, (D) �SMA (E),and zyxin and vinculin mRNA (F) following CG or PBS challenges (d 21 PBS, n5 mice/treatment group; d 21 CG, n5mice/treatment group). Data are expressed as means � se. *P 0.01, **P 0.05.


ciated. Our demonstration that pharmacological inhi-bition of the MRTF-SRF transcriptional activating path-way attenuated peritoneal CTGF expression andfibrosis in the CG model implicates this pathway inperitoneal fibrogenesis. The therapeutic potential oftargeting the MRTF-SRF activating pathway for fibroticdiseases more broadly is underscored by recent studieswith MRTF-A-deficient mice. These mice were dramat-ically protected from cardiac fibrosis produced by ei-ther myocardial infarction or angiotensin II infusion(46). Of note, LPA signaling through LPA1 has recentlybeen reported to induce nuclear translocation of Yes-associated protein (YAP), a major downstream tran-scription factor of the Hippo pathway, through a sig-naling pathway that also involves G�12/13, RhoA, andthe actin cytoskeleton (47). It will be of great interest todescribe the relative roles of SRF- vs. YAP-inducedtranscription in mediating the multiple effects of LPA-LPA1 signaling.

Involvement of G proteins containing G�12/13 sub-units in LPA-induced CTGF expression was demon-strated by significant inhibition of both LPA-inducedRhoA activation and CTGF expression by siRNA knock-down of G�12 and/or G�13. Neither the inhibition ofRhoA activation nor CTGF expression by these siRNAknockdowns was complete, however, suggesting thatother G proteins may be involved in the signalingpathway through which LPA induces CTGF expression.In addition to previously recognized RhoA activation byG�12/13-containing G proteins, G�q-containing G pro-teins have recently been demonstrated to activate RhoAthrough a phospholipase C-Ca2� pathway (48–49),raising the possibility that such G proteins could alsocontribute to LPA-induced CTGF expression. Involve-ment of both MRTF-A and MRTF-B in LPA-inducedCTGF expression was also demonstrated by significant

inhibition of this CTGF expression by siRNA knock-down of either MRTF-A or MRTF-B. Of note, inhibitionof this CTGF expression by MRTF-B knockdown wasgreater than the inhibition produced by MRTF-Aknockdown, despite MRTF-A knockdown being moreefficient than that of MRTF-B. These data suggest thatLPA-induced CTGF expression may be more depen-dent on MRTF-B than MRTF-A.

In addition to the CTGF expression that is inducedby LPA in PMCs being able to drive fibroblast prolifer-ation, fibroblasts themselves have also been reported toproduce CTGF in response to LPA (50), and this CTGFhas been reported to drive fibroblast proliferation in anautocrine fashion (51). Fibroblast proliferation initiallydriven by CTGF produced by PMCs in response to LPAtherefore could be amplified by LPA-induced fibroblastCTGF expression. In addition to its capacity to inducefibroblast proliferation, CTGF directs other importantprofibrotic fibroblast behaviors such as myofibroblastdifferentiation and collagen synthesis (51). We wouldexpect LPA-induced CTGF to contribute to these be-haviors as well, and decreased CTGF-induced myofibro-blast differentiation may also have contributed to thedramatic reduction in peritoneal myofibroblast accu-mulation we observed in CG-challenged LPA1-KO mice.

Finally, our results add yet another tissue type to thegrowing list of tissues and organs in which fibrogenesiscritically depends on the LPA-LPA1 pathway. Includingthis study, data now implicate the LPA-LPA1 pathway inthe development of lung (4), skin (5), kidney (6, 52),liver (53), and peritoneal fibrosis, suggesting that thispathway is of fundamental importance in the pathogen-esis of fibrotic diseases associated with tissue injury. Theaccumulating number of tissues for which fibrosis re-quires LPA-LPA1 signaling suggests that inhibiting thispathway may be broadly beneficial in pathologicalfibrosis. Additionally, by defining a mechanistic linkbetween the profibrotic activities of LPA and CTGF, weconnect 2 important mediators that are currently beingevaluated independently as therapeutic targets in pa-tients with fibrotic diseases.

The authors gratefully acknowledge support from U.S.National Institutes of Health (NIH) grants R01-HL095732and R01-HL108975, and Scleroderma Research Foundation,Pulmonary Fibrosis Foundation, Coalition for PulmonaryFibrosis, and Nirenberg Center for Advanced Lung Diseasegrants to A.M.T, the Japan Society for the Promotion ofScience (JSPS) Excellent Young Researcher Overseas VisitProgram to N.S., and NIH grants R01-MH051699 and R01-DA019674 to J.C. The authors also thank B. A. Fontaine forhis expert assistance, and Amira Pharmaceuticals (San Diego,CA, USA) for the kind gift of AM095. The authors declare noconflicts of interest.

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CTGF

Peritoneal Injury

LPA

Fibroblast CollagenNormalPeritoneum

Gα12/13-Rho-ROCK-actin polymerization-MRTF-SRF pathway

FibroticPeritoneum

LPA1 MesothelilalCells

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Received for publication October 9, 2012.Accepted for publication January 4, 2013.


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