Am J Physiol Lung Cell Mol Physiol 306: L786–L796, 2014. First published February 7, 2014; doi:10.1152/ajplung.00243.2013.

Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor Jibing Yang,1 Miranda Velikoff,1 Ernesto Canalis,2 Jeffrey C. Horowitz,1 and Kevin K. Kim1 1

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; 2Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut Submitted 30 August 2013; accepted in final form 4 February 2014

Yang J, Velikoff M, Canalis E, Horowitz JC, Kim KK. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor. Am J Physiol Lung Cell Mol Physiol 306: L786 –L796, 2014. First published February 7, 2014; doi:10.1152/ajplung.00243.2013.—Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-␤, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-␤-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis. connective tissue growth factor; epithelial-mesenchymal transition; epithelial; fibrosis; lung FIBROSIS OFTEN OCCURS AS A CONSEQUENCE of acute and chronic injury and can occur as a primary process in diseases such as idiopathic pulmonary fibrosis. Progressive fibrosis can lead to organ dysfunction, diminished quality of life, and death. Fibrosis is characterized in part by loss of epithelial cells and accumulation of activated fibroblasts, which deposit excessive fibrotic matrix proteins. Activated fibroblasts also secrete a number of profibrotic factors that recruit and activate more fibrogenic effector cells (1, 2, 49, 56). TGF-␤ is secreted by many cell types and is the most well-established profibrotic factor. TGF-␤ signaling promotes cell transition to an activated fibrogenic phenotype (4). Efforts to target TGF-␤ signaling itself have been proposed as a way to block fibrosis, but this strategy may be limited by the pleotropic effects of TGF-␤, including effects on immunity (17, 40). TGF-␤ induces expression of a number of cytokines with potent fibrogenic effects, most notably, connective tissue growth factor (CTGF). Many

Address for reprint requests and other correspondence: K. Kim, 109 Zina Pitcher Place, BSRB 4061, Ann Arbor, MI 48109 (e-mail: kevkim@med. umich.edu). L786

of the profibrotic effects of TGF-␤ are thought to be mediated through CTGF (12, 29). CTGF is a matricellular protein that is upregulated in many fibrotic tissues. CTGF has been shown to stimulate fibroblast migration, proliferation, and extracellular matrix production in vitro. Inhibition of CTGF activity with neutralizing antibodies attenuates fibrogenesis in animal models and is currently being investigated as a potential therapy for patients with fibrotic disease (27–29, 33). Inhibition of CTGF production might provide an alternative or adjuvant strategy for inhibiting CTGF-mediated fibrogenesis (53). Inhibition of CTGF accumulation during fibrogenesis requires a better understanding of the cell types involved in CTGF production. Unlike TGF-␤ and other profibrotic factors, which are clearly expressed by multiple cellular sources, CTGF expression during fibrogenesis has been suggested as primarily being derived from activated fibroblasts. CTGF is induced by TGF-␤ in a number of fibroblast cell lines, and studies using a CTGF-promoter green fluorescent protein (GFP) transgenic reporter mouse demonstrated CTGF promoter activity exclusively within activated fibroblasts in a model of dermal fibrosis (24). CTGF has thus been utilized as “an effective marker of an activated, fibrotic fibroblast” (27). CTGF expression from epithelial cells and other cell types has been demonstrated in tissues samples from patients with fibrosis and from animal models of fibrosis, but the functional significance of epithelial cell-derived CTGF is not clear (36, 58). The origin of activated fibrogenic cells during fibrosis remains unknown and has been a significant point of controversy. The original model was that expansion and activation of resident fibroblasts fully accounted for the accumulation of fibrogenic effector cells. A second possible source from circulating, bone marrow-derived fibrocytes was identified by their expression of a number of mesenchymal markers (9). Finally, a more recent hypothesis is that structural cells, such as epithelial cells, endothelial cells, and pericytes can respond to injury by changing from a physiological phenotype to a profibrotic phenotype with accompanying changes in protein expression (16, 20, 59). Epithelial-mesenchymal transition (EMT) in vitro and during embryonic development are well established, but EMT during fibrogenesis remains controversial (60). Epithelial cells are abundant in the normal lung, and their function after injury is clearly dynamic, with changes including apoptosis, proliferation, migration, and altered gene expression (50). During injury, epithelial cells may become induced with functions that overlap with important fibrogenic functions of activated fibroblasts, including augmented secretion of profibrotic factors. Prior attempts to identify the contribution of different cell types to fibrogenesis have relied on fate-mapping techniques

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involving mice with a cell type-specific Cre transgene and a lox-stop-lox reporter transgene to specifically and permanently label a population of cells in vivo. However, limitations of the labeling and costaining techniques have led to conflicting reports and continued controversy. At least three independent groups have identified EMT in experimental models of lung fibrosis using this fate-mapping strategy, whereas a recent report found no evidence of EMT in two different lung epithelial cell populations (11, 25, 43, 54). Similarly, there are conflicting reports for EMT in kidney and liver fibrosis (20, 22, 55, 61), pericyte-to-myofibroblast transition in lung fibrosis, and fibrocyte-to-myofibroblast transition during fibrosis (15, 21, 39, 43, 48). Importantly, the fate-mapping reporter genecostaining approach is descriptive without pursing the dynamic functional contribution of different cell types during fibrogenesis and how these changes are regulated. Several recent reports have demonstrated that epithelial cell-specific deletion

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of EMT transcription factors and cell surface receptors can dramatically attenuate fibrosis well beyond the likely proportional contribution of epithelial-derived cells to the pool of activated fibrogenic effector cells, suggesting that activated epithelial cells may mediate recruitment of other fibrogenic effector cells (3, 26, 32, 45). Epithelial cell-specific deletion of secreted fibrotic factors has not been well studied. We recently found that that activated lung epithelial cells can secrete proteins that activate lung fibroblasts, leading to progressive fibrosis. Deletion of type I collagen within lung epithelial cells led to attenuation of lung fibrosis (57). In the present study, we found an important role for epithelial-derived CTGF for activation of fibroblasts and epithelial cells and for lung fibrogenesis. These studies support a model in which epithelial cells can become activated with an early and important role in fibrogenesis through expression of proteins that have traditionally been associated with activated fibroblasts.

Fig. 1. TGF-␤-dependent expression of connective tissue growth factor (CTGF) by primary alveolar epithelial cells (AECs). A–C: primary AECs cultured on fibronectin (FN) have Smad2 phosphorylation and expression of CTGF similar to primary lung fibroblasts stimulated with exogenous TGF-␤1 determined by immunoblot (A) and quantified by densitometry for CTGF (B) and pSmad2 (C). *P ⬍ 0.05 compared with day 0 (D0), n ⫽ 4. D: RT-PCR of AECs cultured on Matrigel (MG), FN, or FN with 10 ␮M SB431542 (FN⫹SB4) for CTGF expression. *P ⬍ 0.05 compared with AECs cultured on FN without inhibitor, n ⫽ 5. E and F: immunoblot of AECs cultured on MG, FN, or FN⫹SB4 for CTGF expression (E) and quantification by densitometry (F). *P ⬍ 0.05 compared with AECs cultured on FN without inhibitor, n ⫽ 3. G: schematic of CTGF conditional-by-inversion (CTGFCOIN) allele. A transmembrane enhanced green fluorescent protein-polyA (GFP pA) sequence is integrated within an intron of the CTGF gene in reverse orientation. The sequence is flanked by lox71 (L71) and lox66 (L66) sequences, enabling Cre recombinase-mediated inversion of the floxed sequence. Cre-mediated inversion yields a CTGFINV allele, in which GFP expression is permanently regulated by the native CTGF promoter, and expression of CTGF itself is permanently blocked by the polyA sequence. H: phase contrast and GFP fluorescence microscopy of AdCre-treated CTGFINV AECs cultured on MG, FN, or FN⫹SB4, ⫻100. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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MATERIALS AND METHODS

Mice. All mice were in a C57bl6 background. CTGF conditionalby-inversion (CTGFCOIN) mice have been previously described (10). Briefly, Cre-mediated inversion yields a CTGFINV allele in which CTGF expression is permanently replaced with GFP reporter expression. Lung epithelial cell-specific inversion of the CTGFCOIN to the CTGFINV allele was achieved by crossing with mice expressing the surfactant protein C (SPC) promoter reverse tetracycline transactivator (SPC-rtTA) and tetO-CMV promoter Cre recombinase (tetO-Cre) transgenes as previously described (25, 26, 38). Breeding mice were maintained on chow supplemented with doxycycline. This system enables broad lung epithelial cell-specific inversion of the CTGFCOIN allele, including airway and alveolar epithelial cells (AECs) of triple transgenic SPC-rtTA/tetO-Cre/CTGFCOIN/COIN (SCctgf) mice. Mice were genotyped by PCR. Littermate control mice lacked at least one component of this triple transgenic system (SPC-rtTA/CTGFCOIN/COIN, tetO-Cre/CTGFCOIN/COIN, CTGFCOIN/COIN, or SPC-rtTA/tetOCre/CTGFWT). Six- to eight-week-old mice were intratracheally injected with 50 ␮l of saline or bleomycin (1.5 U/kg) dissolved in saline. One to three weeks after intratracheal injection, mice were euthanized, and bronchoalveolar lavage (BAL) and lung samples were collected for analysis as previously described (26). Mice were bred and maintained in a specific pathogen-free environment, and all animal experiments were approved by the University Animal Care and Use Committee at the University of Michigan. Reagents. Plasma fibronectin (FN), Y27632, and antibodies to prosurfactant protein-C and phospho-Smad2 are from Millipore. Matrigel was from BD Biosciences. Recombinant TGF-␤1 and TGF-␤1 ELISA Kit are from R&D Systems. Recombinant keratinocyte growth factor is from PeproTech. Small airway growth media (SAGM) is from Lonzo. CTGF antibody and horseradish peroxidaseconjugated secondary antibodies are from Santa Cruz Biotechnology. Immunofluorescent-conjugated secondary antibodies and ProLong Gold Anti-Fade Reagent Containing DAPI are from Life Technologies. Collagen I antibody is from Abcam. SB203580, PD98059, SP600125, LY294002, and antibodies to GFP, phospho-Smad3, and GAPDH are from Cell Signaling. Wortmanin and PF573228 are from Tocris Biosciences. Antibody to Ki67 is from DAKO. Adenoviruses expressing Cre or GFP are from the University of Iowa Gene Transfer Vector Core Facility. All other reagents are from Sigma. Mouse fibroblast and type II cell isolation and culture. Cells were cultured in a 37°C, 5% CO2 incubator. Primary murine lung fibroblasts were isolated from adult mice and cultured as previously described and used between passages 2 and 4 (57). Adult murine lung fibroblast cell line, MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC) and maintained in DMEM with 10% FBS, penicillin, and streptomycin. Murine type II AECs were isolated and cultured in SAGM on tissue culture plates precoated with Matrigel (MG) or FN as previously described (25). In some experiments, cells were maintained in serum-free media and pretreated with chemical inhibitors or vehicle for 2 h and then stimulated with TGF-␤ (4 ng/ml). After an additional 24 h, cells were lysed and analyzed. In some experiments, cells were treated with a daily dose of adenovirus [50 plaque-forming units (pfu)/cell]-expressing GFP or Cre or lentivirus (5 pfu/cell)-encoding siRNA to CTGF or scrambled siRNA for three consecutive days. H&E staining and Masson’s trichrome assay. At the time points indicated, mice were killed, and then lungs were inflated to 25 cmH2O pressure with formaldehyde. Lungs were then paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome by the McClinchey Histology Laboratory (Stockbridge, MI). Hydroxyproline assay. Lung hydroxyproline was measured as previously described (5). Briefly, 3 wk after intratracheal bleomycin injection, lungs were removed and homogenized. Homogenized lungs were incubated in 12 N HCl overnight at 120°C. The samples were

mixed with citrate buffer and chloramine T and then incubated at room temperature for 30 min. Erlich’s solution was then added, and the samples were incubated for an additional 15 min at 65°C. The absorbance at 540 nm was measured, and the hydroxyproline concentration was quantified against hydroxyproline standards. Immunofluorescence staining. After the mice were killed, lungs were inflated with optimal cutting temperature compound. Lungs were then removed and immediately frozen in a dry-ice alcohol bath. Lungs were sectioned to 7 ␮m and were then stained as previously described (25). Briefly, lung sections were fixed with 4% paraformaldehyde and then permeabilized with 1% Triton X-100 in PBS. Lung sections were then blocked in PBS containing 5% normal goat serum and 1% albumin. Primary antibody staining was performed in blocking buffer overnight at 4°C. IgG isotypes were used as negative controls. After lung sections were washed, they were stained with appropriate fluorescent-conjugated secondary antibodies at room temperature for 1 h. Lung sections were then washed and mounted in ProLong Gold containing DAPI. Stained lung sections were visualized using an Olympus BX-51 fluorescence microscope, and images were captured with an Olympus DP-70 camera. Conditioned media stimulation. Primary AECs were cultured on either MG- or FN-coated plates. Some wells were treated with lentivirus-expressing siRNA (5 pfu/cell) or adenovirus-expressing GFP or Cre (50 pfu/cell). Conditioned media (CM) was generated by changing the AEC media to viral-free, serum-free SAGM plus 0.1% BSA 24 to 48 h before collection. The collected CM was filtered to remove cell debris and then either stored at ⫺80°C or immediately added to cultured MLg cell. After 48 h, MLg cells were lysed for various assays. Gene expression analysis. RNA was isolated with TRIzol (Invitrogen) per the manufacturer’s protocol. Reverse transcription was performed with the SuperScript III first-strand synthesis kit (Invitrogen), and RT-PCR was performed using the POWER SYBR Green PCR MasterMix Kit (Applied Biosystems) and Applied Biosystems 7000 sequence detection system. The relative expression levels of genes were normalized to ␤-actin and GAPDH levels as previously described (57). Immunoblot. Immunoblots from cell and lung protein lysates were performed as previously described (25). Scanned immunoblots are representative of at least three independent experiments. Results were quantified by densitometry using NIH ImageJ. TGF-␤1 ELISA. The concentration of total and active TGF-␤ was determined from BAL fluid from control and SCctgf mice at the indicated time points after bleomycin injury using the TGF-␤1 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol. Results are averages of four independent samples quantified against a standard curve. RNA interference assay. The RNA interference vectors were purchased from OpenBiosystems, and lentivirus was generated by the University of Michigan Vector Core. Five pfu per cell of lentivirus

Table 1. Chemical inhibitors to intracellular signaling pathways Inhibitor

Abbreviation

Pathway

SB203580 PD98059 SP600125 Y27632 Wortmanin Ly294002 PF573228 C3 exoenzyme SB431542

SB2 PD SP Y W Ly PF C3 SB4

p38 ERK/MEK JNK Rho PI3 Kinase PI3 Kinase FAK Rho ALK5/TGF-␤R

Final Concentration

10 ␮M 20 ␮M 100 nM 15 ␮M 50 nM 10 ␮M 10 ␮M 1 ␮g/ml 10 ␮M

PI3, phosphatidylinositol 3; FAK, focal adhesion kinase; ALK5, activin receptor-like kinase 5.

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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was used on days 2 and 3 after AEC isolation to inhibit expression of CTGF in primary AECs. Statistical analysis. Data are expressed as means, and error bars indicate SE. For evaluation of group differences, the two-tailed Student’s t-test was used assuming equal variance. A P value of ⬍0.05 was accepted as significant. RESULTS

Primary alveolar epithelial cells express CTGF in vitro. We and others have previously shown that primary AECs upregulate markers of fibrogenic fibroblasts, such as ␣-smooth muscle actin (SMA) and type I collagen, when cultured on FN via autocrine activation of TGF-␤ signaling; in contrast AECs cultured on laminin or MG lack TGF-␤ activation and maintain a type II AEC phenotype (7, 8, 25, 34, 42). We found that CTGF is also highly expressed by AECs under these conditions with levels similar to CTGF expression by lung fibroblasts treated with TGF-␤ for 24 h. Unstimulated lung fibroblasts make very little CTGF (Fig. 1, A and B). The time course of

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CTGF by AECs was similar to the time course for Smad2 phosphorylation, suggesting that CTGF expression was being induced by autocrine TGF-␤ (Fig. 1, A and C). To confirm TGF-␤-dependent expression of CTGF, primary AECs were cultured on MG or on FN in the presence of a TGF-␤ receptor kinase inhibitor (SB431542, 10 ␮M). AECs cultured on MG or with the TGF-␤ inhibitor had less CTGF expression compared with AECs cultured on FN without TGF-␤ inhibition (Fig. 1, D–F). To further confirm TGF-␤-mediated expression of CTGF by primary AECs, we utilized a previously reported conditional-by-inversion CTGFCOIN mouse (10). Briefly, the gene for GFP is embedded within an intron and in reverse orientation allowing normal expression of CTGF. Cre-mediated inversion yields a CTGFINV allele in which GFP expression is regulated by the native CTGF promoter, while CTGF expression is prevented. Thus CTGF expression is permanently deleted within cells expressing Cre, and GFP expression becomes a permanent reporter of native CTGF promoter activity within these cells (Fig. 1G). AECs from CTGFCOIN mice were

Fig. 2. Inhibition of TGF-␤-mediated CTGF expression by primary AECs and primary lung fibroblasts. A: CTGF mRNA expression by lung fibroblasts with or without TGF-␤1 (4 ng/ml) stimulation and treated with intracellular signaling pathway chemical inhibitors or DMSO (DM) vehicle. *P ⬍ 0.05 compared with lung fibroblasts treated with TGF-␤1 and DMSO, n ⫽ 4. B: CTGF mRNA expression by AECs cultured on FN and treated with intracellular signaling pathway chemical inhibitors. *P ⬍ 0.05 compared with AECs treated with DMSO, n ⫽ 4. C: CTGF mRNA expression by primary AECs with or without TGF-␤1 (4 ng/ml) stimulation and treated with intracellular signaling pathway chemical inhibitors or DMSO vehicle. *P ⬍ 0.05 compared with AECs treated with TGF-␤1 and DMSO, n ⫽ 3. D and E: immunoblot for CTGF and phospho-Smad2 (pSmad2) and phospho-Smad3 (pSmad3) by AECs treated with Rho inhibitors and TGF-␤ receptor inhibitor (D) and quantification by immunoblot (E). *P ⬍ 0.01 compared with AECs treated with DMSO, n ⫽ 3. SB2, SB203580; PD, PD98059; SP, SP600125; Y, Y27632; Ly, LY294002; W, Wortmanin; PF, PF573228; SB4, SB431542; C3, C3 exoenzyme. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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isolated, treated with adenovirus-encoding Cre (AdCre) to generate the CTGFINV allele, and cultured on MG or FN. AECs cultured on FN demonstrate marked upregulation of GFP, which is suppressed by TGF-␤ receptor inhibition with SB431542, consistent with TGF-␤-mediated activation of the CTGF promoter within AECs in vitro (Fig. 1H). Because TGF-␤ can activate a number of different intracellular signaling pathways (13), we wanted to determine whether there were differences in the pathways involved in TGF-␤mediated expression of CTGF by fibroblasts and AECs. We used several chemical inhibitors against prominent intracellular signaling pathways (Table 1) to potentially disrupt CTGF expression by fibroblasts stimulated with exogenous TGF-␤ or AECs stimulated by autocrine TGF-␤ activation (Fig. 2). Several inhibitors blocked CTGF expression by both AECs and fibroblasts, including a TGF-␤ receptor kinase inhibitor (SB431542), two phosphatidylinositol 3 kinase inhibitors (Wortmanin and Ly294002), and a focal adhesion kinase inhibitor (PF573228). Inhibition of p38 (SB203580) and ERK (PD98059) MAP kinases partially blocked fibroblast expression of CTGF. In contrast, AECs were very sensitive to a Rho kinase inhibitor, Y27632, which dramatically reduced expression of CTGF by AECs but not fibroblasts. To confirm Rho pathway-dependent expression of CTGF by AECs, we treated primary AECs cultured on FN with SB431542, Y27632, and another Rho inhibitor, exoenzyme C3, and analyzed lysed cells by immunoblot. Consistent with the mRNA data, we found reduction in CTGF protein expression by these three inhibitors. CTGF expression by primary AECs in our system is mediated

by autocrine activation of endogenous TGF-␤ signaling. Rho signaling has been implicated in activation of latent TGF-␤ as well as downstream events after TGF-␤ binding to its receptor (13, 23). To distinguish between these possibilities, the levels of phospho-Smad2 and phospho-Smad3 were determined. We found no difference in Smad2 or Smad3 phosphorylation after treatment with Y27632 or exoenzyme C3, suggesting that the Rho kinase inhibitors are acting primarily on TGF-␤-mediated signaling events downstream of TGF-␤-receptor binding (Fig. 2, D and E). Finally, AECs cultured on FN were further stimulated with exogenously added active TGF-␤ and treated with the panel of chemical inhibitors (Fig. 2C). As expected, the addition of exogenous TGF-␤ only modestly increases CTGF expression, consistent with robust endogenous TGF-␤ activation. Y27632 and SB431542 both dramatically inhibit CTGF expression, confirming the role of these two inhibitors in blocking TGF-␤-mediated induction of CTGF downstream of active TGF-␤ receptor binding. AEC-derived CTGF promotes fibroblast activation. We recently showed that AEC-derived active TGF-␤ remains attached to the extracellular matrix rather than being released into the CM. However, AECs do secrete other TGF-␤-mediated factors that are capable of activating lung fibroblasts in vitro (57). To determine whether AEC-derived CTGF promotes lung fibroblast activation, we used conditioned media from primary AECs from CTGFCOIN mice treated with AdCre or control adenovirus-expressing GFP (AdGFP). Lung fibroblasts treated with TGF-␤ or conditioned media from AdGFPtreated AECs had significant increased expression of type I

Fig. 3. Lung epithelial cell-derived CTGF promotes fibroblast activation. A and B: immunoblot (A) of lung fibroblasts stimulated with AEC-conditioned media (CM) from CTGFCOIN/COIN AECs cultured on FN treated with adenovirus-expressing GFP (AdGFP) or Cre (AdCre) quantified by densitometry (B). AdCre leads to inversion and loss of CTGF expression by CTGFINV/INV AECs and reduced ability to activate lung fibroblast collagen I and ␣-smooth muscle actin (␣-SMA). *P ⬍ 0.05 compared with fibroblasts treated with media and compared with fibroblasts treated with media from AdGFP AECs, n ⫽ 4. C and D: immunoblot of AECs and lung fibroblasts stimulated with AEC-CM (C) quantified by densitometry (D). CM from AECs cultured on FN and treated with no lentivirus (Ctrl CM), lentivirus expressing a scrambled shRNA (shScr CM), and lentivirus expressing shRNA against CTGF (shCTGF CM) were used to stimulate lung fibroblasts. shCTGF inhibits expression of CTGF by AECs (CTGF-AEC). Ctrl CM and shScr CM promote lung fibroblast type I collagen and ␣-SMA expression, which is reduced in fibroblasts treated with shCTGF CM. *P ⬍ 0.05 compared with fibroblasts treated with media and compared with fibroblasts treated with media from shScr AECs, n ⫽ 4. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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collagen and ␣-SMA. In contrast, conditioned media from AdCre-treated AECs resulted in attenuated collagen I and ␣-SMA expression by lung fibroblasts (Fig. 3, A and B). AEC-derived CTGF activation of fibroblasts was confirmed using shRNA directed against CTGF (shCTGF). Again, lung fibroblast treated with TGF-␤, untreated conditioned media, or conditioned media from AECs treated with a control scrambled shRNA (shScr) induced fibroblast expression of type I collagen and ␣-SMA, whereas CM from AECs treated with shCTGF had significantly less induction of collagen I and ␣-SMA expression compared with AECs treated with shScr (Fig. 3, C and D) but more collagen I and ␣-SMA then unstimulated fibroblasts. These results indicate that AEC-derived CTGF significantly participates in fibroblast activation in vitro, but there may be other AEC-derived fibroblast activators. Lung epithelial cell-derived CTGF promotes lung fibrogenesis. To determine whether lung epithelial cell-derived CTGF contributes to fibrogenesis, CTGFCOIN/COIN mice were crossed with SPC-rtTA, tetO-CMV-Cre mice. Breeders were maintained on doxycycline to generate mice in which CTGF is

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permanently deleted and GFP is permanently expressed by the native CTGF promoter specifically within lung epithelialderived cells, including airway and AECs (Fig. 4A). These triple transgenic mice will be subsequently referred to as SCctgf. Littermate mice lacking at least one of the transgenes were used as controls. Cultured AECs from SCctgf mice had significantly less CTGF expression and robust GFP expression compared with AECs from littermate control mice lacking one of the transgenes that had high CTGF expression and no GFP expression (Fig. 4B). AECs from SCctgf mice also exhibited decreased levels of collagen I and ␣-SMA, indicating a critical role for CTGF in AEC EMT in vitro (Fig. 4C). Primary lung fibroblasts from SCctgf and littermate control mice had similar levels of CTGF in response to TGF-␤ and no GFP expression (Fig. 4D). These results confirm robust and lung epithelial-specific deletion of CTGF expression and replacement with CTGF promoterdriven GFP expression in SCctgf mice. Lung sections from uninjured SCctgf mice were histologically similar to uninjured control mouse lung (Fig. 4, E and F), indicating that

Fig. 4. Generation and validation of mice with lung epithelial cell-specific deletion of CTGF. A: lung epithelial cell-specific and permanent deletion of CTGF is achieved using transgenic mice carrying the surfactant proteins-C (SPC) promoter-reverse tetracycline transactivator (SPC-rtTA) and tetO-CMV promoter-Cre recombinase (tetO-Cre). In triple transgenic mice (SCctgf), the SPC promoter yields rtTA expression specifically within lung epithelial cells. In the presence of doxycycline (dox), rtTA activates the tetO-CMV promoter, leading to expression of Cre recombinase and inversion of the CTGFCOIN allele. B: AECs from SCctgf mice have diminished expression of CTGF and induced expression of GFP by immunoblot compared with AECs from littermate control mice lacking 1 of the 3 transgenes that have robust expression of CTGF and absent expression of GFP. Differences in CTGF expression were quantified by immunoblot. *P ⬍ 0.05 compared with control, n ⫽ 3. C: AECs from SCctgf mice cultured on FN have diminished expression of collagen I, ␣-SMA, and CTGF. Differences were quantified by immunoblot. *P ⬍ 0.05 compared with control, n ⫽ 3. D: primary lung fibroblasts from SCctgf mice have similar CTGF expression compared with control lung fibroblasts, verifying robust and lung epithelial-specific replacement of CTGF expression with GFP. Differences were quantified by immunoblot. Control and SCctgf are not statistically different, n ⫽ 4. E and F: uninjured lungs from SCctgf (F) mice have normal histology compared with littermate genotype control mice (E), hematoxylin and eosin (H&E) (⫻200). AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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lung epithelial cell-derived CTGF is not necessary for normal lung development. SCctgf and littermate control mice were injured with intratracheal saline or bleomycin. One week after bleomycin injury, SCctgf and littermate control lungs were analyzed by immunoblot and immunofluorescence staining (Fig. 5). Littermate control mice exhibited no GFP expression, whereas SCctgf mice exhibited marked upregulation of GFP expression, indicating activation of CTGF promoter within lung epithelialderived cells in vivo in response to bleomycin. The GFP staining was noted within AECs, airway epithelial cells, and periairway cells, suggesting that CTGF production by lung epithelial cells is not limited to AECs but likely includes airway epithelial cells and their derivatives. Some of these cells were undergoing proliferation as indicated by Ki67 staining. Furthermore, costained lung sections indicated that not all SPC-positive cells become GFP-positive after bleomycin injury. One week after bleomycin, there was a similar increase in the extent of injury/inflammation between SCctgf and control mice determined by BAL cell count and protein (Fig. 6). In contrast, 3 wk after bleomycin, SCctgf mice had significantly less fibrosis compared with littermate genotype control mice as

visualized by lung sections stained with H&E and trichrome (Fig. 7). Attenuation in collagen accumulation was quantified by hydroxyproline, which again indicated attenuation in fibrosis in SCctgf mice compared with littermate control mice, confirming the importance of lung epithelial cell-derived CTGF to fibrogenesis (Fig. 7E). Analysis of SCctgf and control mice indicated preserved early production and activation of TGF-␤ with subsequent attenuation in TGF-␤ and myofibroblast accumulation, indicated by ␣-SMA expression, at 3 wk after bleomycin in SCctgf mice (Fig. 8). DISCUSSION

These findings support a model in which lung epithelial cells can become activated and acquire functions that overlap known important functions of activated fibroblasts during fibrogenesis. We found that lung epithelial cells produce CTGF in vitro and in vivo and demonstrate the importance of lung epithelial cell-derived CTGF to fibrogenesis. Inhibition or deletion of CTGF can attenuate fibrogenesis although, to our knowledge, there is presently no report on the impact of CTGF deletion on lung fibrosis and no report on the deletion of epithelial cell-

Fig. 5. Activation of lung epithelial cell CTGF expression acutely after bleomycin (Bleo) injury. A–D: GFP immunofluorescence staining of lung sections (⫻200). A: uninjured SPC-rtTA/CTGFCOIN/COIN genotype control mouse, lacking the tetO-Cre transgene. B: uninjured SCctgf mouse. C: tetO-Cre/CTGFCOIN/COIN genotype control mouse, lacking the SPC-rtTA transgene, 1 wk after bleomycin. D: SCctgf mouse, 1 wk after bleomycin. SCctgf mice injured with bleomycin have significant periairway and alveolar staining for GFP, indicating activation of the CTGF promoter from cells of epithelial origin. E: IgG isotype control for GFP staining from SCctgf mouse 1 wk after bleomycin (⫻200). F: lung section from SCctgf mouse 1 wk after bleomycin stained for GFP (green) and prosurfactant protein-C (red) (⫻400). G: lung section from SCctgf mouse 1 wk after bleomycin stained for GFP (green) and Ki67 (red) (⫻400). H and I: immunoblot (H) of whole lung lysate from littermate genotype control and SCctgf mice uninjured or 1 wk after bleomycin. Uninjured SCctgf lungs have weak expression of GFP and upregulation of GFP after bleomycin, whereas control mice have no expression of GFP. Differences were quantified by densitometry (I). *P ⬍ 0.05 compared with lungs from SCctgf mice injured with bleomycin. **P ⬍ 0.01 compared with uninjured SCctgf mice, n ⫽ 3. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00243.2013 • www.ajplung.org

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Fig. 6. Acute injury after bleomycin is independent of lung epithelial CTGF. Bronchoalveolar lavage (BAL) from control and SCctgf mice 1 wk after bleomycin or saline injury analyzed for total cell count (A) and protein (B). SCctgf mice and control mice have similar increases in proteins and cell count 1 wk after bleomycin. n ⫽ 4 – 6 per group.

specific deletion of CTGF on fibrosis. Deletion of CTGF within AECs resulted in attenuation of bleomycin-induced lung fibrosis and impaired autocrine and paracrine type collagen I expression. Indeed CTGF has been implicated in EMT and fibroblast activation (27, 28, 52). Activated fibrogenic cells secrete profibrotic factors in addition to ECM proteins propagating progressive fibrogenesis. In tissue with an abundance of epithelial cells, as with the distal lung, initiation of fibrogenesis may be dependent on activation of these prominent resident

structural cells, leading to recruitment and activation of other cell types such as fibroblasts and fibrocytes in addition to autocrine activation of AECs. Prior studies evaluating the potential role of different cell types as fibrogenic effector cells during fibrosis have utilized a fate-mapping system in which fibrotic tissue is costained for the cell-specific reporter and for markers of activated fibroblasts. The design of the CTGFCOIN allele permits an “all-inone” fate-mapping strategy that overcomes many of the limi-

Fig. 7. Lung epithelial cell-derived CTGF promotes lung fibrogenesis. A: lung sections from littermate control (left) and SCctgf (right) mice 3 wk after bleomycin injection stained with H&E (top) and trichrome (bottom) ⫻100. Genotype control mice have robust fibrosis compared with SCctgf mice. E: hydroxyproline assay from entire lungs 3 wk after saline or bleomycin in SCctgf or littermate control mice. SCctgf mice have less fibrosis after bleomycin (n ⫽ 4 – 8 per group), *P ⬍ 0.05 compared with control mice treated with bleomycin.

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Fig. 8. Expression of CTGF, ␣-SMA, and TGF-␤ at 1-wk intervals after bleomycin injury. A: whole lung lysate from SCctgf and littermate control mice were analyzed for expression of ␣-SMA and CTGF. B: densitometry quantification of ␣-SMA immunoblot. *P ⬍ 0.05 compared with control mice at day 21, n ⫽ 3. C: densitometry quantification of CTGF immunoblot. *P ⬍ 0.05 compared with control mice at day 7, n ⫽ 3. D and E: ELISA for active TGF-␤ (D) and total (E) from BAL of control and SCctgf mice. *P ⬍ 0.05 compared with control mice at day 14, n ⫽ 3– 6.

tations of the costaining approach. In this system, GFP expression indicates a cell that is both of lung epithelial cell origin and has activation of the native CTGF promoter, thus eliminating the potential ambiguities of dual staining. Whether CTGF expression is a sufficient marker to define EMT is unclear, but CTGF is clearly an important profibrotic cytokine whose expression has been associated with activated fibrogenic effector cells. Inhibition of CTGF expression did not completely abrogate fibroblast activation by activated AECs, suggesting the presence of other fibroblast-activating factors. Epithelial-mesenchymal interaction through a variety of mediators is emerging as an important regulator of lung fibrosis (18). We recently identified a number of profibrotic-secreted factors highly expressed by activated AECs, including type I collagen, type III collagen, and thrombospondin, which may potentially be involved in fibroblast activation (57). Using a similar approach, we recently found that lung epithelial cells significantly contribute to the accumulation of type I collagen, an important fibrotic matrix protein that has been thought to be primarily derived from activated fibroblasts (57). Future stud-

ies using this approach could assess the functional contribution of collagen I and CTGF from other cell types and epithelial cell-specific contribution of other secreted proteins typically associated with activated fibroblasts. Cosuppression of several of these AEC-derived factors or identification and inhibition of a transcriptional program regulating the expression of many of these factors is likely to have a more dramatic effect on fibrogenesis. We found that AEC expression of CTGF is induced by TGF-␤, similar to fibroblasts. The numerous different intracellular signaling pathways activated by TGF-␤ are highly dependent on the cell type (13). We used a panel of inhibitors to target prominent intracellular signaling pathways known to be induced by TGF-␤. We found that expression of CTGF by primary AECs was particularly sensitive to inhibition of Rho signaling. We previously reported dramatic differences in cell shape when culturing primary AECs on different matrices, suggesting a potential role for actin cytoskeletal regulation of transcription mediated through the Rho pathway (25, 35). A number of studies have suggested that signaling through Rho

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family members is critical for epithelial cell acquisition of mesenchymal features, and inhibition of Rho kinase has been shown to attenuate fibrosis in several animal models (19, 37, 41, 51). Our model of AEC induction of CTGF utilizes autocrine production, activation, and response to TGF-␤. Rho kinase has been implicated both in the activation of latent TGF-␤ and during the intracellular signaling that occurs after active TGF-␤ binds to its cell surface receptor. In our system, we found that Smad2/3 phosphorylation was not altered by Rho kinase inhibition, indicating preserved activation of latent TGF-␤, suggesting that Rho activates CTGF expression downstream of Smad2/3 phosphorylation or via a Smad-independent pathway (13, 23). A number of different mediators have linked Rho signaling to TGF-␤-induced expression of CTGF (6, 31, 35, 46). We are actively investigating which of these mediators are involved in AECs. Importantly, these studies confirm that there are significant differences between fibroblast and AEC production of CTGF. Our in vivo system enabled inversion of CTGFCOIN allele within lung epithelial cells broadly. We found dramatic expression of GFP within airway epithelial cells after bleomycin injury, suggesting that expression of CTGF by lung epithelial cells is not limited to AECs and that functional differences likely extend to different subtypes of lung fibroblasts and epithelial cells, as others have previously suggested (14, 30, 47). For our in vivo studies, our initial approach has been to target lung epithelial cells broadly, but comparing the functional differences among lung epithelial cell populations will be important for future studies. However, the traditional classification of lung epithelial cell subtypes likely does not capture the true functional heterogeneity among lung epithelial cells (30, 44). Differences in relevant intracellular signaling pathways between fibroblasts and lung epithelial cells are likely not limited to the ones identified in this study. A more complete understanding of the contributions and regulation of different cell types to progressive fibrogenesis may lead to new targets for potential therapeutic intervention. ACKNOWLEDGMENTS The authors thank Kelly McDonough and Zhen Geng for technical assistance. GRANTS This work was supported by National Institute of Health (NIH) grant R01 HL108904 (K. Kim), K08 HL085290 (K. Kim), and R01 AR021707 (E. Canalis). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: J.Y., E.C., J.C.H., and K.K.K. conception and design of research; J.Y., M.V., and K.K.K. performed experiments; J.Y., M.V., and K.K.K. analyzed data; J.Y., M.V., E.C., J.C.H., and K.K.K. interpreted results of experiments; J.Y., M.V., and K.K.K. prepared figures; J.Y., M.V., and K.K.K. drafted manuscript; J.Y., M.V., E.C., J.C.H., and K.K.K. edited and revised manuscript; J.Y., M.V., E.C., J.C.H., and K.K.K. approved final version of manuscript. REFERENCES 1. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment International consensus statement American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med 161: 646 –664, 2000.

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