Loss of PPARγ expression by fibroblasts enhances dermal wound closure
© Sha et al; licensee BioMed Central Ltd. 2012
Received: 4 January 2012
Accepted: 13 April 2012
Published: 13 April 2012
Peroxisome proliferator-activated receptor (PPAR)γ may be a key regulator of connective tissue deposition and remodeling in vivo. PPARγ expression is reduced in dermal fibroblasts isolated from fibrotic areas of scleroderma patients; PPARγ agonists suppress the persistent fibrotic phenotype of this cell type. Previously, we showed that loss of PPARγ expression in fibroblasts resulted in enhanced bleomycin-induced skin fibrosis. However, whether loss of PPARγ expression in skin fibroblasts affects cutaneous tissue repair or homeostasis is unknown.
Mice deleted for PPARγ in skin fibroblasts show an enhanced rate of dermal wound closure, concomitant with elevated phosphorylation of Smad3, Akt and ERK, and increased expression of proliferating cell nuclear antigen (PCNA), collagen, α-smooth muscle actin (α-SMA) and CCN2. Conversely, dermal homeostasis was not appreciably affected by loss of PPARγ expression.
PPARγ expression by fibroblasts suppresses cutaneous tissue repair. In the future, direct PPARγ antagonists and agonists might be of clinical benefit in controlling chronic wounds or scarring, respectively.
If the dermis is injured, specialized fibroblasts called myofibroblasts repopulate the wound and synthesize and remodel new connective tissue . Wound repair is very complex and dynamic, involving the interactions of multiple cell types and growth factors/cytokines; dysregulation of this process results in chronic wounds or fibrosis . Thus, understanding the molecular mechanisms controlling the normal tissue repair program is likely to be of clinical relevance.
Expression of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ is reduced in dermal fibroblasts isolated from fibrotic lesions of patients with the autoimmune connective tissue disease scleroderma (systemic sclerosis, SSc); moreover, the PPARγ agonist rosiglitazone reverses the persistent fibrotic phenotype of this cell type . Normally, PPAR-γ is bound to the retinoid X receptor (RXR) and co-repressors, preventing its binding to DNA; however, upon receptor ligation, the co-repressors are displaced from the PPAR-γ/RXR complex allowing PPAR-γ to bind PPAR-γ response elements in the promoters of target genes . The PPAR/RXR transcriptional complex plays a critical role in maintaining energy balance, which is dysregulated in conditions such as obesity, diabetes, and atherosclerosis .
An increasing body of evidence also suggests that PPAR-γ plays a key role in connective tissue turnover, a key process involved with tissue remodeling . Both in vivo and in vitro, PPAR-γ agonists inhibit basal and transforming growth factor (TGF)-β-induced collagen deposition and myofibroblast differentiation [6, 7]. Although loss of PPARγ expression in cultured mouse embryonic fibroblasts results in enhanced, constitutive Smad3 phosphorylation and collagen production , loss of PPARγ expression in cultured adult mouse fibroblasts appears to be insufficient to result in either Smad 3 activation or collagen production . Instead, adult dermal fibroblasts lacking PPARγ expression show enhanced sensitivity to exogenously added TGFβ in terms of enhanced phosphorylation of Smad3 and expression of collagen/α-SMA mRNA . Collectively, these data strongly suggest that PPARγ may play a role in fibrosis by enhancing cellular responses to TGFβ.
Recently, we showed that mice harboring a fibroblast-specific deletion for PPARγ displayed an enhanced susceptibility to bleomycin-induced skin scleroderma . These observations suggest that PPARγ might be an important regulator of cutaneous tissue repair and homeostasis in vivo; however, this hypothesis has yet to be tested. Herein, we subject mice harboring a fibroblast-specific deletion of PPARγ to the dermal punch model of cutaneous tissue repair. Moreover, we assess whether loss of PPARγ expression by skin fibroblasts affects dermal homeostasis. Our results reveal new insights into the role PPARγ plays in fibroblast biology.
Loss of PPARγ expression in skin results in faster wound closure
Loss of PPARγ does not appreciably affect dermal homeostasis
Collectively, our results indicate that, in vivo, PPARγ normally acts in dermal fibroblasts to suppress fibrogenic responses.
In this study, we tested the effect of loss of PPARγ in dermal fibroblasts on cutaneous tissue repair in vivo. PPARγ-deficient mice showed enhanced responsiveness to tissue injury, as shown by increased wound closure on days three, five, seven and ten post-wounding, increased collagen production, the appearance of α-SMA-expressing myofibroblasts, elevated CCN2 expression and enhanced Smad3/Akt phosphorylation. These results are consistent with previous observations that PPARγ-deficient fibroblasts showed enhanced sensitivity to TGFβ1 and that PPARγ-deficient mice showed increased susceptibility to bleomycin-induced skin fibrosis [8, 9]. These data also agree with prior studies showing that, in fibroblasts, PPARγ agonists block TGFβ-induced α-SMA and collagen expression . Moreover, we showed that, although CCN2 mRNA was elevated in PPARγ-knockout fibroblasts, troglitazone suppressed CCN2 mRNA in both wild-type and PPARγ-knockout fibroblasts. Although it is possible that these PPARγ agonists were active due to the small residual expression of PPARγ in PPARγ knockout fibroblasts, these data do not exclude the notion that thiazolidinediones such as troglitazone have potent 'off-target' effects independent of PPARγ itself . Nonetheless, these data collectively suggest that PPARγ normally suppresses fibrogenic responses in vivo and also support the notion that developing novel classes of direct PPARγ agonists/antagonists is warranted.
We found that long-term (up to four months) deletion of PPARγ did not appreciably affect dermal homeostasis. This result is consistent with our previous data showing that mice deficient in PPARγ expression in fibroblasts (approximately six weeks post-deletion of PPARγ), although possessing enhanced susceptibility to bleomycin-induced skin fibrosis, possessed no detectable alterations in skin structure . PPARγ is known to upregulate the tumor suppressor PTEN; loss of PTEN expression by dermal fibroblasts results in skin fibrosis due to an increase in Akt phosphorylation [13, 18]. Intriguingly, we found that PTEN expression was increased and not decreased in the dermis of PPARγ knockout mice (data not shown). These observations suggest that PPARγ knockout mice may upregulate PTEN expression to compensate for the loss of PPARγ expression by dermal fibroblasts and provide a plausible explanation why dermal homeostasis was not appreciably altered in PPARγ knockout mice and injury, whether caused by bleomycin or by dermal punch wounding, is required to elicit a phenotype in PPARγ knockout animals.
In summary, our studies examining the involvement in PPARγ in skin indicate that PPARγ normally acts in fibroblasts to retard tissue repair. These results suggest that direct PPARγ agonists and antagonists might be used to control the tissue repair program, for example, by suppressing scarring or by promoting the closure of chronic wounds.
Generation of PPARγ conditional knockout mice
Mice, hemizygous for an allele expressing tamoxifen-inducible Cre-recombinase (under the control of a fibroblast-specific regulatory sequence from the proa2(I) collagen gene ) and homozygous for a loxP-PPARγ allele, were generated as described previously . Mice deleted (K/K) or not (C/C) for PPARγ in fibroblasts were generated by treating three-week-old mice each day for five days with tamoxifen (4-hydroxitamoxifen, Sigma, St. Louis, MO, USA) in corn oil (0.1 ml of 10 mg/ml) or corn oil. Deletion of PPARγ was verified by PCR genotyping (Jackson Laboratories, Bar Harbor, ME, USA).γ All animal protocols were approved by the regulatory authority of the appropriate experimental animal committee.
Cell culture, immunofluorescence and Western analysis
Dermal fibroblasts, isolated from explants (four- to six-week-old animals) as described, were subjected to indirect immunofluorescence analysis followed by an appropriate secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) as described . Photography (Zeiss Axiphot) was performed using a digital camera (Empix, Mississauga, ON, Canada). For some assays, cells were lysed in 2% SDS, proteins quantified (Fisher, Nepean, ON, Canada) and subjected to Western blot analysis . Antibodies used were: anti-a-SMA (Sigma,1:3000), anti-β-actin (1:5000, Sigma) and anti- PPARγ (Santa Cruz, Santa Cruz, CA, USA,1:500).γ Human dermal fibroblasts were purchased (American Type Culture Collection, Manassas, VA, USA). Cells were grown in (D)MEM, 10% fetal bovine serum (Life Technologies, Burlington, ON, Canada).
Assessment of collagen content
To assess the effects of PPARγ deletion on collagen synthesis, trichrome collagen stain was used. Collagen content in each section was assessed by three blinded observers using the following assessment criteria: 0 signifies: no collagen fibers; 1 signifies: few collagen fibers; 2 signifies: moderate amount of collagen fibers; 3 signifies: excessive amount of collagen fibers.
Non-specific immunoglobulin G (IgG) binding was blocked by incubating sections with BSA (0.1%) in PBS for 1 hour and then incubated with primary antibody in a humidified chamber and left overnight at 4°C. Next, sections were washed and incubated with a secondary antibody for 1 hour. Primary antibodies used were: PPARγ (Santa Cruz, Rabbit, sc-7196, 1:500) PTEN (Cell Signaling, Pickering, ON, Canada, #9559, Rabbit, 1:500) CCN2 (Abcam, Cambridge, MA, USA, ab6992, Rabbit, 1:250), α-SMA (Sigma, A5228, mouse, 1:2000); ColIa2 (Santa Cruz, sc-28654, rabbit, 1:500), PCNA: (Abcam, ab2426-1, rabbit, 1:500); p-SMAD3: (Abcam, rabbit, ab52903, 1:200); p-AKT (Rockland Immunochemicals, Gilbertsville, PA, USA, 200-301-268, mouse), (1:500); p-ERK (Cell Signaling, #9101, rabbit, 1:500) and neutrophil marker (Santa Cruz, sc-59338, rat,1:100). Secondary antibodies (Jackson Immunoresearch, 1:400) were: Dylight 488 conjugated donkey anti-mouse (711-485-150); Dylight 594 conjugated donkey anti-rabbit (711-485-152); Dylight 594 conjugated donkey anti-mouse (715-515-150) and Dylight 594 conjugated donkey anti-rat (712-516-150).
Real-time PCR to detect the expression of target genes was performed essentially as previously described [20, 21]. Cells were cultured until 50% confluence and treated for 24 hours with dimethyl sulfoxide (DMSO) or troglitazone (40 μM, EMD Biosciences, Billerica, MA, USA) and total RNA was isolated (RNeasy; QIAGEN, Toronto, ON, Canada). Total RNA (25 ng) was reverse transcribed, amplified using Taq-Man Assays-on-Demand in the presence One-Step MasterMix and detected using the ABI Prism 7900 HT sequence detector (Life Technologies). Triplicate samples were run, and expression values for CCN2 were standardized to values obtained with control 18S primers using the ΔΔCt method.
Statistical analysis was done using one way analysis of variance (ANOVA) and Tukey's post-hoc test on Graphpad Prism 4 software.
Bovine serum albumin
(Dulbecco's) modified Eagle's medium
- H & E:
Hematoxylin and eosin
Proliferating cell nuclear antigen
Polymerase chain reaction
Peroxisome proliferator-activated receptorγ
Retinoid X receptor
α-smooth muscle actin
Transforming growth factor-β.
This work is supported by grants from the Canadian Institutes of Health Research (to AL and MB) and the Ontario Scleroderma Society (to AL). AL is a member of the Canadian Scleroderma Research Group New Emerging Team. WS and KT were the recipients of salary awards from the Canadian Scleroderma Research Group.
- Tomasek JJ, Gabbiani G, Hinz B, Brown RA: Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2003, 3: 349-363.View ArticleGoogle Scholar
- Elliott CG, Hamilton DW: Deconstructing fibrosis research: do pro-fibrotic signals point the way for chronic dermal wound regeneration?. J Cell Commun Signal. 2011, 5: 301-315. 10.1007/s12079-011-0131-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Shi-wen X, Eastwood M, Stratton RJ, Denton CP, Leask A, Abraham DJ: Rosiglitazone alleviates the persistent fibrotic phenotype of lesional skin scleroderma fibroblasts. Rheumatology (Oxford). 2010, 49: 259-263. 10.1093/rheumatology/kep371.View ArticleGoogle Scholar
- Plutzky J: The PPAR-RXR transcriptional complex in the vasculature: energy in the balance. Circ Res. 2011, 108: 1002-1016. 10.1161/CIRCRESAHA.110.226860.View ArticlePubMedGoogle Scholar
- Wei J, Bhattacharyya S, Varga J: Peroxisome proliferator-activated receptor γ innate protection from excessive fibrogenesis and potential therapeutic target in systemic sclerosis. Curr Opin Rheumatol. 2010, 22: 671-676. 10.1097/BOR.0b013e32833de1a7.PubMed CentralView ArticlePubMedGoogle Scholar
- Burgess HA, Daugherty LE, Thatcher TH, Lakatos HF, Ray DM, Redonnet M, Phipps RP, Sime PJ: PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 2005, 288: L1146-L1153. 10.1152/ajplung.00383.2004.View ArticlePubMedGoogle Scholar
- Wu M, Melichian DS, Chang E, Warner-Blankenship M, Ghosh AK, Varga J: Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma. Am J Pathol. 2009, 174: 519-533. 10.2353/ajpath.2009.080574.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghosh AK, Wei J, Wu M, Varga J: Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-gamma. Biochem Biophys Res Commun. 2008, 374: 231-236. 10.1016/j.bbrc.2008.07.014.PubMed CentralView ArticlePubMedGoogle Scholar
- Kapoor M, McCann M, Liu S, Huh K, Denton CP, Abraham DJ, Leask A: Loss of PPARγ in mouse fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis. Arthritis Rheum. 2009, 60: 2822-2829. 10.1002/art.24761.View ArticlePubMedGoogle Scholar
- Shi-wen X, Stanton LA, Kennedy L, Pala D, Chen Y, Howat SL, Renzoni EA, Carter DE, Bou-Gharios G, Stratton RJ, Pearson JD, Beier F, Lyons KM, Black CM, Abraham DJ, Leask A: CCN2 is necessary for adhesive responses to transforming growth factor-beta1 in embryonic fibroblasts. J Biol Chem. 2006, 281: 10715-10726. 10.1074/jbc.M511343200.View ArticlePubMedGoogle Scholar
- Kulkarni AA, Thatcher TH, Olsen KC, Maggirwar SB, Phipps RP, Sime PJ: PPAR-γ ligands repress TGFβ-induced myofibroblast differentiation by targeting the PI3K/Akt pathway: implications for therapy of fibrosis. PLoS One. 2011, 6: e15909-10.1371/journal.pone.0015909.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu SW, Liu S, Eastwood M, Sonnylal S, Denton CP, Abraham DJ, Leask A: Rac inhibition reverses the phenotype of fibrotic fibroblasts. PLoS One. 2009, 4: e7438-10.1371/journal.pone.0007438.View ArticlePubMedGoogle Scholar
- Parapuram SK, Shi-wen X, Elliott C, Welch ID, Jones H, Baron M, Denton CP, Abraham DJ, Leask A: Loss of PTEN expression by dermal fibroblasts causes skin fibrosis. J Invest Dermatol. 2011, 131: 1996-2003. 10.1038/jid.2011.156.View ArticlePubMedGoogle Scholar
- Huang W, András IE, Rha GB, Hennig B, Toborek M: PPARα and PPARγ protect against HIV-1-induced MMP-9 overexpression via caveolae-associated ERK and Akt signaling. FASEB J. 2011, 25: 3979-3988. 10.1096/fj.11-188607.PubMed CentralView ArticlePubMedGoogle Scholar
- Leask A, Holmes A, Black CM, Abraham DJ: Connective tissue growth factor gene regulation. Requirements for its induction by transforming growth factor-beta 2 in fibroblasts. J Biol Chem. 2003, 278: 13008-13015. 10.1074/jbc.M210366200.View ArticlePubMedGoogle Scholar
- Zhang M, Fraser D, Phillips A: ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-beta1 autoinduction in proximal tubular epithelial cells. Am J Pathol. 2006, 169: 1282-1293. 10.2353/ajpath.2006.050921.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Beek JP, Kennedy L, Rockel JS, Bernier SM, Leask A: The induction of CCN2 by TGFbeta1 involves Ets-1. Arthritis Res Ther. 2006, 8: R36-10.1186/ar1890.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang W, Wu N, Li Z, Wang L, Jin J, Zha XL: PPARgamma activator rosiglitazone inhibits cell migration via upregulation of PTEN in human hepatocarcinoma cell line BEL-7404. Cancer Biol Ther. 2006, 5: 1008-1014. 10.4161/cbt.5.8.2887.View ArticlePubMedGoogle Scholar
- De Val S, Ponticos M, Antoniv TT, Wells DJ, Abraham D, Partridge T, Bou-Gharios G: Identification of the key regions within the mouse pro-alpha 2(I) collagen gene far-upstream enhancer. J Biol Chem. 2002, 277: 9286-9292. 10.1074/jbc.M111040200.View ArticlePubMedGoogle Scholar
- Fajardo OA, Thompson K, Parapuram SK, Liu S, Leask A: Mithramycin reduces expression of fibro-proliferative mRNAs in human gingival fibroblasts. Cell Prolif. 2011, 44: 166-173. 10.1111/j.1365-2184.2011.00738.x.View ArticlePubMedGoogle Scholar
- Thompson K, Hamilton DW, Leask A: ALK5 inhibition blocks TGFβ-induced CCN2 expression in gingival fibroblasts. J Dent Res. 2010, 89: 1450-1454. 10.1177/0022034510379020.View ArticlePubMedGoogle Scholar
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