Impact of high-/middle-molecular-weight adiponectin on the synthesis and regulation of extracellular matrix in dermal fibroblasts
2014; Elsevier BV; Volume: 42; Issue: 4 Linguagem: Inglês
10.1016/j.exphem.2013.12.009
ISSN1873-2399
AutoresHideki Nakasone, Kiriko Terasako‐Saito, Rie Yamazaki, Miki Sato, Yukië Tanaka, Kana Sakamoto, Masakazu Kurita, Ryoko Yamasaki, Hidenori Wada, Yuko Ishihara, Koji Kawamura, Tomohito Machishima, Masahiro Ashizawa, Shun-ichi Kimura, Misato Kikuchi, Aki Tanihara, Junya Kanda, Shinichi Kako, Junji Nishida, Shigeki Yamada, Yoshinobu Kanda,
Tópico(s)Skin and Cellular Biology Research
ResumoAdiponectin has been shown to play a critical role in immunity. Recently, we reported that the adiponectin levels after allogeneic stem cell transplantation were higher in recipients with chronic graft-versus-host disease (cGVHD). However, the effects of adiponectin on extracellular matrix (ECM) and regulatory factors in dermal fibroblasts remain unclear. We compared the messenger RNA (mRNA) levels of collagen type1 (COL1A), fibronectin 1 (FN1), matrix metalloproteinase (MMP)1, MMP3, tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, transforming growth factor-β (TGF-β), and TGF-β receptor 2 (TGF-βR2) in human normal dermal fibroblasts cultured with and without adiponectin, and we assessed the degree of synthesis of ECMs by immunofluorescent microscopy. Furthermore, we also assessed these mRNA levels after blocking of TGF-βR2. Adiponectin induced higher mRNA levels of FN1, MMP1, MMP3, TIMP1, TIMP3, and TGF-βR2 in a dose-dependent manner, but did not significantly affect COL1A or TGF-β. In addition, adiponectin was shown to upregulate FN1, MMPs, and TIMPs after blocking of TGF-βR2. Immunofluorescent microscopy revealed that adiponectin promoted a greater synthesis of ECMs than in the control in vitro. The finding that adiponectin upregulated ECM-associated factors might mean that high levels of adiponectin could modulate dermal fibrosis was observed in recipients with cGVHD. Further basic investigation is warranted to elucidate whether the adiponectin-pathway could be a target for the treatment of sclerotic cGVHD. Adiponectin has been shown to play a critical role in immunity. Recently, we reported that the adiponectin levels after allogeneic stem cell transplantation were higher in recipients with chronic graft-versus-host disease (cGVHD). However, the effects of adiponectin on extracellular matrix (ECM) and regulatory factors in dermal fibroblasts remain unclear. We compared the messenger RNA (mRNA) levels of collagen type1 (COL1A), fibronectin 1 (FN1), matrix metalloproteinase (MMP)1, MMP3, tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, transforming growth factor-β (TGF-β), and TGF-β receptor 2 (TGF-βR2) in human normal dermal fibroblasts cultured with and without adiponectin, and we assessed the degree of synthesis of ECMs by immunofluorescent microscopy. Furthermore, we also assessed these mRNA levels after blocking of TGF-βR2. Adiponectin induced higher mRNA levels of FN1, MMP1, MMP3, TIMP1, TIMP3, and TGF-βR2 in a dose-dependent manner, but did not significantly affect COL1A or TGF-β. In addition, adiponectin was shown to upregulate FN1, MMPs, and TIMPs after blocking of TGF-βR2. Immunofluorescent microscopy revealed that adiponectin promoted a greater synthesis of ECMs than in the control in vitro. The finding that adiponectin upregulated ECM-associated factors might mean that high levels of adiponectin could modulate dermal fibrosis was observed in recipients with cGVHD. Further basic investigation is warranted to elucidate whether the adiponectin-pathway could be a target for the treatment of sclerotic cGVHD. Allogeneic stem cell transplantation (SCT) is an important curative treatment for hematologic diseases. However, SCT is associated with many adverse complications, including graft-versus-host disease (GVHD). GVHD is thought to be the result of alloreactive and autoreactive interactions among donor T and B cells, host antigen-presenting cells, and host tissues [1Paczesny S. Hanauer D. Sun Y. Reddy P. New perspectives on the biology of acute GVHD.Bone Marrow Transplant. 2010; 45: 1-11Crossref PubMed Scopus (129) Google Scholar, 2Sarantopoulos S. Stevenson K.E. Kim H.T. et al.Altered B-cell homeostasis and excess BAFF in human chronic graft-versus-host disease.Blood. 2009; 113: 3865-3874Crossref PubMed Scopus (245) Google Scholar, 3Shimabukuro-Vornhagen A. Hallek M.J. Storb R.F. von Bergwelt-Baildon M.S. The role of B cells in the pathogenesis of graft-versus-host disease.Blood. 2009; 114: 4919-4927Crossref PubMed Scopus (208) Google Scholar]. In particular, chronic GVHD (cGVHD) significantly impairs the recipient's quality of life [4Ferrara J.L. Levine J.E. Reddy P. Holler E. Graft-versus-host disease.Lancet. 2009; 373: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar, 5Pidala J. Anasetti C. Jim H. Quality of life after allogeneic hematopoietic cell transplantation.Blood. 2009; 114: 7-19Crossref PubMed Scopus (231) Google Scholar]. The detailed mechanism of cGVHD has not been elucidated, although previous reports have investigated various biomarkers for cGVHD [6Schultz K.R. Miklos D.B. Fowler D. et al.Toward biomarkers for chronic graft-versus-host disease: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: III. Biomarker Working Group Report.Biol Blood Marrow Transplant. 2006; 12: 126-137Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. Almost all these biomarkers were shown to be inflammatory cytokines, such as tumor necrosis factor α, soluble interleukin-2 receptor, and soluble B cell activation factor, which are associated with the activation or inhibition of immune cells, including T and B cells [2Sarantopoulos S. Stevenson K.E. Kim H.T. et al.Altered B-cell homeostasis and excess BAFF in human chronic graft-versus-host disease.Blood. 2009; 113: 3865-3874Crossref PubMed Scopus (245) Google Scholar, 7Fujii H. Cuvelier G. She K. et al.Biomarkers in newly diagnosed pediatric-extensive chronic graft-versus-host disease: a report from the Children's Oncology Group.Blood. 2008; 111: 3276-3285Crossref PubMed Scopus (118) Google Scholar, 8Imamura M. Hashino S. Kobayashi H. et al.Serum cytokine levels in bone marrow transplantation: synergistic interaction of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha in graft-versus-host disease.Bone Marrow Transplant. 1994; 13: 745-751PubMed Google Scholar, 9Liem L.M. van Houwelingen H.C. Goulmy E. Serum cytokine levels after HLA-identical bone marrow transplantation.Transplantation. 1998; 66: 863-871Crossref PubMed Scopus (72) Google Scholar]. However, few reports have focused on other endocrine substances in the view of pathophysiology of cGVHD. Recently, it has been revealed that adiponectin, an adipokine that is secreted by adipose tissues, plays an important role in immunity and inflammation [10Yamauchi T. Kadowaki T. Physiological and pathophysiological roles of adiponectin and adiponectin receptors in the integrated regulation of metabolic and cardiovascular diseases.Int J Obes (Lond). 2008; 32: S13-S18Crossref PubMed Scopus (282) Google Scholar, 11Stofkova A. Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity.Endocr Regul. 2009; 43: 157-168PubMed Google Scholar, 12Fantuzzi G. Adiponectin and inflammation: consensus and controversy.J Allergy Clin Immunol. 2008; 121: 326-330Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 13Tilg H. Moschen A.R. Role of adiponectin and PBEF/visfatin as regulators of inflammation: involvement in obesity-associated diseases.Clin Sci (Lond). 2008; 114: 275-288Crossref PubMed Scopus (204) Google Scholar, 14Otero M. Lago R. Gomez R. et al.Changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis.Ann Rheum Dis. 2006; 65: 1198-1201Crossref PubMed Scopus (463) Google Scholar, 15Yamamoto K. Kiyohara T. Murayama Y. et al.Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease.Gut. 2005; 54: 789-796Crossref PubMed Scopus (208) Google Scholar]. Adiponectin is thought to exist in a globular isoform, trimers, and middle-/high-molecular-weight (MMW/HMW-) multimers. AdipoR1, AdipoR2, and T-cadherin have been identified as specific receptors for each, respectively [16Brochu-Gaudreau K. Rehfeldt C. Blouin R. Bordignon V. Murphy B.D. Palin M.F. Adiponectin action from head to toe.Endocrine. 2010; 37: 11-32Crossref PubMed Scopus (246) Google Scholar]. The functions of adiponectin are diverse and may depend on the target organ and its isoforms [16Brochu-Gaudreau K. Rehfeldt C. Blouin R. Bordignon V. Murphy B.D. Palin M.F. Adiponectin action from head to toe.Endocrine. 2010; 37: 11-32Crossref PubMed Scopus (246) Google Scholar, 17Neumann E. Frommer K.W. Vasile M. Muller-Ladner U. Adipocytokines as driving forces in rheumatoid arthritis and related inflammatory diseases?.Arthritis Rheum. 2011; 63: 1159-1169Crossref PubMed Scopus (85) Google Scholar]. We recently reported that high levels of HMW-adiponectin were observed after SCT in recipients who suffered from cGVHD, and were associated with the severity of cGVHD [18Nakasone H. Binh P.N. Yamazaki R. et al.Association between serum high-molecular-weight adiponectin level and the severity of chronic graft-versus-host disease in allogeneic stem cell transplantation recipients.Blood. 2011; 117: 3469-3472Crossref PubMed Scopus (13) Google Scholar]. However, it is still unclear whether the increase in HMW-adiponectin is a primary or secondary event, as is the role that HMW-adiponectin plays in the pathophysiology of cGVHD. We hypothesized that HMW-adiponectin might have fibrotic or antifibrotic effects on dermal fibroblasts, because skin fibrosis is a major symptom of cGVHD. Skin and organ fibrosis are both defined as the excessive deposition and accumulation of extracellular matrix (ECM), including collagen type 1 and fibronectin [19Jinnin M. Mechanisms of skin fibrosis in systemic sclerosis.J Dermatol. 2010; 37: 11-25Crossref PubMed Scopus (126) Google Scholar]. This ECM is produced by dermal fibroblasts, and is known to be increased in scleroderma [20Cotton S.A. Herrick A.L. Jayson M.I. Freemont A.J. TGF beta–a role in systemic sclerosis?.J Pathol. 1998; 184: 4-6Crossref PubMed Scopus (63) Google Scholar]. The ECMs produced by fibroblasts are regulated by matrix metalloproteinase (MMP), which can degrade ECMs, and by tissue inhibitor of metalloproteinase (TIMP), which can inhibit the activity of MMPs. Therefore, MMPs might improve fibrosis, whereas TIMPs might accelerate the deposition of ECMs and fibrosis [19Jinnin M. Mechanisms of skin fibrosis in systemic sclerosis.J Dermatol. 2010; 37: 11-25Crossref PubMed Scopus (126) Google Scholar]. Both MMPs and TIMPs are also produced by fibroblasts. To date, there has no thorough investigation of the effects of HMW-/MMW-adiponectin on ECM, MMPs, and TIMPs in dermal fibroblasts. Therefore, we assessed the changes in the gene expression of ECMs and regulatory factors, including transforming growth factor (TGF), MMPs and TIMPs, with or without MMW-/HMW-adiponectin in normal dermal fibroblasts in vitro. A skin sample was obtained during plastic surgery with informed consent. Superficial dermal samples were incubated with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) for 16–24 hours at 4°C, and the epithelium was separated from the superficial dermal sample. Next, human fibroblasts were isolated and cultured for explant at 37°C under a humidified atmosphere of 5% CO2 in fibroblast growth medium including Dulbecco's modified Eagle's medium with 10% fetal calf serum and 0.6 mg/mL glutamine. Approximately 3 weeks later, primary cultures were subcultured. These dermal fibroblasts derived from a normal subject were used for all experiments during 7–12 passages. Human recombinant HMW-/MMW-oligomer-rich adiponectin was purchased commercially (BioVendor, Asheville, NC, USA). Fibroblasts were cultured in a humidified atmosphere of 5% CO2 at 37°C until subconfluence, and harvested with 0.025% trypsin and 0.01% EDTA. At day −1, 5,000 fibroblasts/cm2 were seeded in each well of an IWAKI 24-well plate in M106 medium (Kurabo, Osaka, Japan) containing 2% fetal bovine serum with 10 μg/mL gentamicin and 0.25 μg/mL amphotericin. After 24 hours (at day 0), each well was washed with PBS and exchanged for 0.5 mL of fresh M106 medium with or without HMW-/MMW-adiponectin. For the time-dependent assessment, 0 or 10 μg/ml of HMW-/MMW-adiponectin was added to control and target wells, respectively. Fibroblasts were collected at 0, 24, 48, and 72 hours after the addition of adiponectin. For the dose-dependent assessment, 0, 1, 5, 10, or 20 μg/mL of HMW-/MMW-adiponectin was added to each well. Fibroblasts were collected 3 days after the addition of adiponectin. The adiponectin level in normal subjects is considered to range between 2 and 10 μg/ml [11Stofkova A. Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity.Endocr Regul. 2009; 43: 157-168PubMed Google Scholar, 16Brochu-Gaudreau K. Rehfeldt C. Blouin R. Bordignon V. Murphy B.D. Palin M.F. Adiponectin action from head to toe.Endocrine. 2010; 37: 11-32Crossref PubMed Scopus (246) Google Scholar]. Furthermore, we compared the gene expression of ECMs, MMPs, and TIMPs under TGF-β receptor 2 (TGF-βR2)-blocked conditions using 20 μg/ml of anti-human TGF-βR2 antibody (R&D Systems, Minneapolis, MN, USA) and 20 μg/ml of HMW-/MMW-adiponectin. A dose of 10–20 μg/mL of antihuman TGF-βR2 antibody has often been used for the neutralization of TGF-pathways [21Tsang M.L. Zhou L. Zheng B.L. et al.Characterization of recombinant soluble human transforming growth factor-beta receptor type II (rhTGF-beta sRII).Cytokine. 1995; 7: 389-397Crossref PubMed Scopus (73) Google Scholar]. In the same manner as described earlier, fibroblasts were collected 3 days after cytokine administration. The targets and controls each included two or three wells. After fibroblasts were collected, messenger RNA (mRNA) extraction and complementary DNA (cDNA) synthesis were performed using an RNAspin mini RNA isolation kit (GE Healthcare, Tokyo, Japan) and SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies, Tokyo, Japan) according to the respective manufacturer's instructions. We then performed quantitative real-time reverse-transcript polymerase chain reaction (qRT-PCR) using Taqman Universal Master Mix II (Life Technologies, Tokyo, Japan) according to the manufacturer's instructions. All specific primers and probes for targets and internal control genes were purchased from Life Technologies (Tokyo, Japan): T-cadherin (Hs00169908_m1*), fibronectin 1 (FN1) (Hs01549976_m1*), collagen type I alpha 2 (COL1A2) (Hs00164099_m1*), TIMP-1 (Hs00171558_m1*), TIMP-3 (Hs00165949_m1*), MMP-1 (Hs00899658_m1*), MMP-3 (Hs00968305_m1*), TGF-β1 (Hs99999918_m1), and TGF-βR2 (Hs00234253_m1*). β-actin (433762F) was used as an internal control. All qRT-PCR procedures were performed with a 7900HT FAST Real Time PCR system (Life Technologies, Tokyo, Japan). Relative transcripts were determined by the following formula: 2–(CT target – CT control). Observations of ECMs in vitro were performed by immunofluorescent microscopy as described in previous reports [22Lareu R.R. Subramhanya K.H. Peng Y. et al.Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.FEBS Lett. 2007; 581: 2709-2714Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 23Chen C.Z. Peng Y.X. Wang Z.B. et al.The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well.Br J Pharmacol. 2009; 158: 1196-1209Crossref PubMed Scopus (113) Google Scholar, 24Karamichos D. Guo X.Q. Hutcheon A.E. Zieske J.D. Human corneal fibrosis: an in vitro model.Invest Ophthalmol Vis Sci. 2010; 51: 1382-1388Crossref PubMed Scopus (116) Google Scholar]. At day −1, 5,000 fibroblasts/cm2 were seeded on cover glasses in IWAKI 35 mm dishes with M106 medium (Kurabo, Osaka, Japan) containing 2% FBS. After 24 hours (day 0), each dish was washed with PBS and exchanged for fresh M106 medium without any cytokines, with 20 ng/mL of TGF-β or 20 μg/mL of HMW-/MMW-adiponectin. At day 4, cells were washed with PBS and fixed with 3.3% formaldehyde of CellFIX (BD Biosciences, Tokyo, Japan) for 15 minutes and 0.5% triton X (Nacalai Tesque, Kyoto, Japan) for 5 minutes. After three washes with PBS, the samples were blocked with PBS containing 2% FBS for 30 minutes. After three washes with PBS, the samples were incubated with primary antibodies of anti-fibronectin IgG produced in rabbit (1:200 in PBS; F3648; Sigma, Tokyo, Japan) and anti-collagen type1 IgG produced in mouse (1:1000 in PBS; C2456; Sigma) at 4°C for 4 hours. After three washes with PBS, anti-rabbit IgG produced in chicken AlexaFluor488 (A21441; Life Technology) and anti-mouse IgG produced in donkey AlexaFluor594 (A21203; Life Technologies) were added (1:500 in PBS for each) and incubated for 30 minutes. ProLong Gold Antifade Reagent with 4′,6- diamidino-2-phenylindoldilactate (Life Technologies, Tokyo, Japan) was added to each sample after three washes with PBS. Images were obtained by laser confocal microscopy (Fluoview Systems FV500; Olympus, Tokyo, Japan). Immunohistochemistry (IHC) analyses for fibronectin, MMP-1, MMP-3, TIMP-1, TIMP-3, and TGF-βR2 were performed using formalin-fixed, paraffin-embedded skin samples of a healthy subject and a patient with cGVHD of the skin. In addition, we examined monocyte chemotactic protein-1 (MCP-1) as a representative of proinflammatory cytokines, because it is known to be induced by adiponectin in autoimmune arthritis [11Stofkova A. Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity.Endocr Regul. 2009; 43: 157-168PubMed Google Scholar, 12Fantuzzi G. Adiponectin and inflammation: consensus and controversy.J Allergy Clin Immunol. 2008; 121: 326-330Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar]. The samples of a healthy subject were purchased commercially from ILSBio (Chestertown, MD, USA). The skin samples of cGVHD-involved and noninvolved regions were obtained from an autopsy of a patient with skin cGVHD who received steroid administration for 3 years for GVHD and finally died of sepsis-induced thrombotic thrombocytopenic purpura. Sections (4 μm each) were deparaffinized with xylene and ethanol. Next, the sections were treated with 0.125% trypsin at 37°C for 10 minutes for fibronectin, high pH 9.0 in Tris-EDTA solutions at 97°C for 40 minutes for MMP-3 and TGF-βR2, and low pH 6.0 in citric acid solutions at 97°C for 40 minutes for MMP-1, TIMP-1, TIMP-3, and MCP-1, respectively. Next, they were incubated with primary antibodies at room temperature for 30 minutes in the following dilutions: anti-fibronectin IgG produced in rabbit (1:800, F3648; Sigma), anti–MMP-1 IgG produced in rabbit (1:400, GTX100534; Genetex, Irvine, CA, USA), anti–MMP-3 IgG produced in rabbit (1:1000, GTX100723; Genetex), anti–TIMP-1 IgG produced in mouse (1:200, MS-606-p0; LVC, Fremont, CA, USA), anti–TIMP-3 IgG produced in rabbit (1:400, LLC250885; Abbiotec, San Diego, CA, USA), anti–TGF-βR2 IgG produced in rabbit (1:400, LLC250880; Abbiotec), and anti–MCP-1 IgG produced in rabbit (1:400, 500-P34; Peprotech Rocky Hill, NJ, USA). Thereafter, using EnVision Flex detection system (DAKO, Tokyo, Japan) with Autostainer Link48 (DAKO), the secondary reactions were performed according to the manufacturer's instructions. After peroxidase blocking for 5 minutes, the sections were treated by EnVision FLEX polymer (DAKO). The nuclei were stained with hematoxylin. These views were obtained with Nano Zoomer 2.0RS (Hamamatsu Photonics, Hamamatsu, Japan) and NDP.view2 (Hamamatsu Photonics). Student t test and analysis of variance followed by post hoc Tukey multiple comparisons were used for comparisons of mRNA expression in fibroblasts. In addition, the Jonckheere–Terpstra test was used to assess dose dependency. Statistical significance was defined as a two-tailed p < 0.05. All statistical analyses were performed with EZR (Saitama Medical Centre, Jichi Medical University; http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html) [44Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics.Bone Marrow Transplant. 2013; 48: 452-458Crossref PubMed Scopus (8346) Google Scholar], which is a graphical user interface for R (The R Foundation for Statistical Computing, version 2.13.0). More precisely, it is a modified version of R commander (version 1.6-3) that was designed to add statistical functions that are frequently used in biostatistics. This study was approved by the institutional review board of Jichi Medical University. The expression of T-cadherin, the receptor of HMW-/MMW-adiponectin, is known to differ according to the target organ [25Ivanov D. Philippova M. Antropova J. et al.Expression of cell adhesion molecule T-cadherin in the human vasculature.Histochem Cell Biol. 2001; 115: 231-242PubMed Google Scholar]. Therefore, we first confirmed that T-cadherin is expressed in human dermal fibroblasts (Fig. 1A and B). The expression of T-cadherin at 48 and 72 hours after HMW-/MMW-adiponectin administration was higher than that in controls (Fig. 1A), and this effect was dose dependent (P < 0.0001, Jonckheere–Terpstra test; Fig. 1B). Next, we assessed the effect of HMW-/MMW-adiponectin on gene expression for FN1 and COL1A2. The expression of FN1 was 2.0-, 1.8-, and 4.0-fold higher than that in controls at 24, 48, and 72 hours after administration, respectively (Fig. 2A). The effect of HMW-/MMW-adiponectin on the expression of FN1 was dose dependent: 1.4-, 3.0-, 4.7-, and 7.8-fold higher than that in the control for fibroblasts cultured with 1, 5, 10, and 20 μg/mL of adiponectin, respectively (p < 0.001, Jonckheere–Terpstra test; Fig. 2B). On the other hand, there was no difference in the expression of COL1A2 between fibroblasts with and without HMW-/MMW-adiponectin (Fig. 2C and D). Among metalloproteinase families, we measured the gene expression of MMP-1, MMP-3, TIMP-1, and TIMP-3 because the expression of MMP-1, MMP-3 and TIMP-1 is known to be associated with dermal fibrosis in patients with systemic sclerosis [26Kuroda K. Shinkai H. Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis.Arch Dermatol Res. 1997; 289: 567-572Crossref PubMed Scopus (88) Google Scholar]. In addition, TIMP-1 and TIMP-3 are highly expressed in skin involvement of GVHD [27Salmela M.T. Karjalainen-Lindsberg M.L. Jeskanen L. Saarialho-Kere U. Overexpression of tissue inhibitor of metalloproteinases-3 in intestinal and cutaneous lesions of graft-versus-host disease.Mod Pathol. 2003; 16: 108-114Crossref PubMed Scopus (14) Google Scholar]. The expression of MMP-1 in fibroblasts with HMW-/MMW-adiponectin increased in a time-dependent manner, whereas MMP-1 expression in fibroblasts without HMW-/MMW-adiponectin increased 24 hours after administration and then decreased (Fig. 3A). The expression of MMP-1 was dose-dependently higher than that in controls: 5.1-, 7.0-, 15.4-, and 32.6-fold higher in wells with 1, 5, 10, and 20 μg/mL adiponectin, respectively (p < 0.001, Jonckheere–Terpstra test; Fig. 3B). The effect of HMW-/MMW-adiponectin on the expression of MMP-3 in fibroblasts became apparent at 48 hours or later; 1.2-, 2.2-, and 5.1-fold higher than that in controls at 24, 48, and 72 hours after administration, respectively (Fig. 3C), and this effect was dose dependent (p < 0.001, Jonckheere–Terpstra test; Fig. 3D).Figure 3Comparisons of the relative transcripts of metalloproteinase families. Comparisons of the expression of metalloproteinase families evaluated by quantitative real time polymerase chain reaction for (A, B) MMP-1, (C, D) MMP-3, (E, F) TIMP-1, and (G, H) TIMP-3—(A, C, E, G) in a time-dependent manner at 0, 24, 48, and 72 hours after 0 or 10 μg/mL of high- or middle-molecular-weight adiponectin administration and (B, D, F, H) in a dose-dependent manner 3 days after adiponectin administration (0, 1, 5, 10, or 20 μg/mL). The comparisons are shown between target and control cells. *p < 0.05; **p < 0.01; ***p < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of HMW-/MMW-adiponectin on the expression of TIMP-1 in fibroblasts was similar to that on the expression of MMP-1 and MMP-3, with an apparent dose-dependent effect at 48 hours or later (Fig. 3E and F). In contrast, the effect of HMW-/MMW-adiponectin on the expression of TIMP-3 appeared earlier: 1.8-, 3.1-, and 2.3-fold higher than that in controls at 24, 48, and 72 hours after administration, respectively, although the total expression started to decrease 48 hours after administration in wells with and without adiponectin (Fig. 3G). The effect of HMW-/MMW-adiponectin on the expression of TIMP-3 was also dose dependent (p < 0.001, Jonckheere–Terpstra test; Fig. 3H). Based on our observations, we assessed whether HMW-/MMW-adiponectin could produce or degrade ECMs in vitro. Under the current experimental setting, HMW-/MMW-adiponectin seemed to induce greater synthesis and deposition of both fibronectin and collagen type 1 than that in the control, and the effect seemed to be comparable to that of TGF-β1 (Fig. 4). Next, we assessed whether HMW-/MMW-adiponectin would produce TGF-β1 and TGF-βR2, a well-known fibrogenic cytokine and receptor. There was no significant difference in the expression of TGF-β1 between fibroblasts with and without HMW-/MMW-adiponectin in this experimental setting (Fig. 5A and B). On the other hand, the expression of TGF-βR2 was 1.9- and 1.6-fold higher than that in the control group at 48 and 72 hours after administration, respectively (Fig. 5C). The expression of TGF-βR2 was dose-dependently higher than that in the control group; 1.6-, 1.8-, 2.2-, and 3.4-fold in wells with 1, 5, 10, and 20 μg/mL of HMW-/MMW-adiponectin, respectively (p < 0.001, Jonckheere–Terpstra test; Fig. 5D). Next, we assessed whether the effect of HMW-/MMW-adiponectin was dependent on a TGF-β pathway under TGF-βR2–blocked conditions. The increased expression of ECMs, MMPs, and TIMPs by HMW-/MMW-adiponectin was not suppressed by the addition of anti–TGF-βR2 antibody (Fig. 6A–F). Immunohistochemistry was performed for samples of a normal subject, cGVHD-involved and noninvolved skin region of a patient with skin cGVHD. A diffuse increase of fibronectin expression with strong staining was observed in dermis of the involved region of skin cGVHD compared with normal skin and noninvolved region of skin cGVHD (Fig. 7). TGF-βR2, MMP-3, and MCP-1 were stained mainly in ducts and endothelial cells. An increase of spindle cells that express TGF-βR2 (strong staining), MMP-3 (relatively weak staining), and MCP-1 (weak and small amount) were observed especially in papillary dermis of the involved region of skin cGVHD compared with normal skin and noninvolved region of skin cGVHD (Fig. 7). In addition, they were increased in epidermis of both involved and noninvolved skin regions of the patient compared with normal skin. Cells with MMP-1 expression were also observed in the dermis of the involved region of skin cGVHD, although they were few in number and sporadic with a weak staining (Fig. 7). On the other hand, we could not find any differences in the expressions of TIMP-1 and TIMP-3. Adiponectin has been shown to have both proinflammatory and anti-inflammatory functions [12Fantuzzi G. Adiponectin and inflammation: consensus and controversy.J Allergy Clin Immunol. 2008; 121: 326-330Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar]. In obesity-related diseases such as diabetes mellitus, adiponectin is thought to induce IL-10, and to have anti-inflammatory effects and protect against cardiovascular events [10Yamauchi T. Kadowaki T. Physiological and pathophysiological roles of adiponectin and adiponectin receptors in the integrated regulation of metabolic and cardiovascular diseases.Int J Obes (Lond). 2008; 32: S13-S18Crossref PubMed Scopus (282) Google Scholar, 11Stofkova A. Leptin and adiponectin: from energy and metabolic dysbalance to inflammation and autoimmunity.Endocr Regul. 2009; 43: 157-168PubMed Google Scholar]. On the other hand, high adiponectin levels have been observed and associated with the disease severity in autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and diabetes mellitus type 1 [15Yamamoto K. Kiyohara T. Murayama Y. et al.Production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in Crohn's disease.Gut. 2005; 54: 789-796Crossref PubMed Scopus (208) Google Scholar, 17Neumann E. Frommer K.W. Vasile M. Muller-Ladner U. Adipocytokines as driving forces in rheumatoid arthritis and related inflammatory diseases?.Arthritis Rheum. 2011; 63: 1159-1169Crossref PubMed Scopus (85) Google Scholar, 28Ebina K. Fukuhara A. Ando W. et al.Serum adiponectin concentrations correlate with severity of rheumatoid arthritis evaluated by extent of joint destruction.Clin Rheumatol. 2009; 28: 445-451Crossref PubMed Scopus (81) Google Scholar, 29Hadjadj S. Aubert R. 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Although there have been few reports on systemic sclerosis, high adiponectin levels were positively correlated to disease duration and a high skin-thickness score [33Masui Y. Asano Y. Shibata S. et al.Serum adiponectin levels inversely correlate with the activity of progressive skin sclerosis in patients with diffuse cutaneous systemic sclerosis.J Eur Acad Dermatol Venereol. 2011; 26: 354-360Crossref PubMed Scopus (45) Google Scholar]. In addition, it has been shown that the skin of nonobese people, who are thought to have higher adiponectin levels, is thicker than that of obese people [34Smalls L.K. Randall Wickett R. Visscher M.O. Effect of dermal thickness, tissue composition, and body site on skin biomechanical properties.Skin Res Technol. 2006; 12: 43-49Crossref PubMed Scopus (140) Google Scholar]. The controversy regarding whether adiponectin has a proinflammatory or anti- inflammatory effect might be due to the fact that most observations and animal models have been based on the use of mixtures of all of the isoforms of adiponectin. Recently, it has been suggested that adiponectin might have different effects on different target cells and tissues, and that its functions might be different according to its isoforms [16Brochu-Gaudreau K. Rehfeldt C. Blouin R. Bordignon V. Murphy B.D. Palin M.F. Adiponectin action from head to toe.Endocrine. 2010; 37: 11-32Crossref PubMed Scopus (246) Google Scholar, 17Neumann E. Frommer K.W. Vasile M. Muller-Ladner U. Adipocytokines as driving forces in rheumatoid arthritis and related inflammatory diseases?.Arthritis Rheum. 2011; 63: 1159-1169Crossref PubMed Scopus (85) Google Scholar]. Therefore, we used only HMW-/MMW-adiponectin in our experiments. In addition, no previous study has assessed the effect of adiponectin on not only ECM but also both MMPs and TIMPs in human dermal fibroblasts (Table 1) [35Wanninger J. Walter R. Bauer S. et al.MMP-9 activity is increased by adiponectin in primary human hepatocytes but even negatively correlates with serum adiponectin in a rodent model of non-alcoholic steatohepatitis.Exp Mol Pathol. 2011; 91: 603-607Crossref PubMed Scopus (23) Google Scholar, 36Essick E.E. Ouchi N. Wilson R.M. et al.Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling.Am J Physiol Heart Circ Physiol. 2011; 301: H984-H993Crossref PubMed Scopus (87) Google Scholar, 37Kang E.H. Lee Y.J. Kim T.K. et al.Adiponectin is a potential catabolic mediator in osteoarthritis cartilage.Arthritis Res Ther. 2010; 12: R231Crossref PubMed Scopus (107) Google Scholar, 38Benaitreau D. Dos Santos E. Leneveu M.C. et al.Effects of adiponectin on human trophoblast invasion.J Endocrinol. 2010; 207: 45-53Crossref PubMed Scopus (45) Google Scholar, 39Handy J.A. Saxena N.K. Fu P. et al.Adiponectin activation of AMPK disrupts leptin-mediated hepatic fibrosis via suppressors of cytokine signaling (SOCS-3).J Cell Biochem. 2010; 110: 1195-1207Crossref PubMed Scopus (78) Google Scholar, 40Lago R. Gomez R. Otero M. et al.A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes.Osteoarthritis Cartilage. 2008; 16: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 41Chen T.H. Chen L. Hsieh M.S. Chang C.P. Chou D.T. Tsai S.H. Evidence for a protective role for adiponectin in osteoarthritis.Biochim Biophys Acta. 2006; 1762: 711-718Crossref PubMed Scopus (171) Google Scholar, 42Kumada M. Kihara S. Ouchi N. et al.Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages.Circulation. 2004; 109: 2046-2049Crossref PubMed Scopus (464) Google Scholar]. Therefore, we assessed the effects of HMW-/MMW-adiponectin on human dermal fibroblasts.Table 1Summary of other investigations that have assessed the effects of adiponectin on both MMPs and TIMPsaData from http://www.ncbi.nlm.nih.gov/pubmed/, using the search terms adiponectin, MMP, and TIMP.Adiponectin isoformTarget cellMatrix metalloproteinase (MMP)Tissue inhibitor of metalloproteinase (TIMP)ReferenceFull-length adiponectin, trimers—MMP-9 gene expression ↑TIMP-1 expression ↑35Wanninger J. Walter R. Bauer S. et al.MMP-9 activity is increased by adiponectin in primary human hepatocytes but even negatively correlates with serum adiponectin in a rodent model of non-alcoholic steatohepatitis.Exp Mol Pathol. 2011; 91: 603-607Crossref PubMed Scopus (23) Google ScholarIn vivo study using knockout vs. wild type miceRat and mouse cardiomyocyteROS-induced MMP-2 and MMP-9 activity ↓MMP-2-to-TIMP-2 and MMP-9-to-TIMP-1 ratios ↑ in knockout mice36Essick E.E. Ouchi N. Wilson R.M. et al.Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling.Am J Physiol Heart Circ Physiol. 2011; 301: H984-H993Crossref PubMed Scopus (87) Google ScholarFull-length adiponectin, trimersHuman chondrocytes of osteoarthritisMMP-1, MMP-3, and MMP-13 expression and secretion ↑TIMP-1 expression, no change37Kang E.H. Lee Y.J. Kim T.K. et al.Adiponectin is a potential catabolic mediator in osteoarthritis cartilage.Arthritis Res Ther. 2010; 12: R231Crossref PubMed Scopus (107) Google ScholarNo detailsHuman trophoblastMMP-2 and MMP-9 activity ↑TIMP-1 expression, no change38Benaitreau D. Dos Santos E. Leneveu M.C. et al.Effects of adiponectin on human trophoblast invasion.J Endocrinol. 2010; 207: 45-53Crossref PubMed Scopus (45) Google ScholarTIMP-2 expression ↓No detailsRat hepatic stellate cellsMMP-1 activity ↑Leptin-stimulated TIMP-1 ↓39Handy J.A. Saxena N.K. Fu P. et al.Adiponectin activation of AMPK disrupts leptin-mediated hepatic fibrosis via suppressors of cytokine signaling (SOCS-3).J Cell Biochem. 2010; 110: 1195-1207Crossref PubMed Scopus (78) Google ScholarFull-length adiponectin, TrimersHuman and murine chondrocytesMMP-3 and MMP-9 secretion ↑TIMP-1 secretion, no change40Lago R. Gomez R. Otero M. et al.A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes.Osteoarthritis Cartilage. 2008; 16: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (238) Google ScholarMMP-2 secretion, no changeFull-length adiponectin, trimersHuman chondrocyteIL-β–induced MMP-13 expression ↑TIMP-2 expression ↑41Chen T.H. Chen L. Hsieh M.S. Chang C.P. Chou D.T. Tsai S.H. Evidence for a protective role for adiponectin in osteoarthritis.Biochim Biophys Acta. 2006; 1762: 711-718Crossref PubMed Scopus (171) Google ScholarMMP-3 expression, no changeTIMP-1 expression, no changeNo detailsHuman monocyte-derived macrophagesMMP-9 expression, no changeTIMP-1 expression ↑42Kumada M. Kihara S. Ouchi N. et al.Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages.Circulation. 2004; 109: 2046-2049Crossref PubMed Scopus (464) Google Scholara Data from http://www.ncbi.nlm.nih.gov/pubmed/, using the search terms adiponectin, MMP, and TIMP. Open table in a new tab The current study showed that HMW-/MMW-adiponectin induced higher gene expression and synthesis of FN1 in dermal fibroblasts. On the other hand, HMW-/MMW-adiponectin did not affect the gene expression of COL1A2, but did induce the greater deposition of collagen type 1 than in the control, which is consistent with a previous report that adiponectin upregulates the secretions but not the gene expression of collagen [43Ezure T. Amano S. Adiponectin and leptin up-regulate extracellular matrix production by dermal fibroblasts.Biofactors. 2007; 31: 229-236Crossref PubMed Scopus (55) Google Scholar]. It is well known that TGF-β1 is associated with the accumulation of ECMs and fibrosis [19Jinnin M. Mechanisms of skin fibrosis in systemic sclerosis.J Dermatol. 2010; 37: 11-25Crossref PubMed Scopus (126) Google Scholar, 20Cotton S.A. Herrick A.L. Jayson M.I. Freemont A.J. TGF beta–a role in systemic sclerosis?.J Pathol. 1998; 184: 4-6Crossref PubMed Scopus (63) Google Scholar]. HMW-/MMW-adiponectin did not have a significant effect on the expression of TGF-β1. On the other hand, it significantly upregulated the expression of TGF-βR2. However, the promoting effects of HMW-/MMW-adiponectin on ECM expression were not suppressed by the neutralization of TGF-βR2. Therefore, HWM-/MMW-adiponectin can increase the expression of ECM independent from the TGF-βR2 pathways. HMW-/MMW-adiponectin not only induced the synthesis and deposition of ECMs; it also upregulated the expression of both TIMPs and MMPs. These findings are consistent with the observation that TIMP-1, MMP-1, and MMP-3 are all increased in dermal fibroblasts in the early stages of systemic sclerosis, whereas MMP-1 and MMP-3 are decreased in the late stages [26Kuroda K. Shinkai H. Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis.Arch Dermatol Res. 1997; 289: 567-572Crossref PubMed Scopus (88) Google Scholar]. Taken together, these findings suggest that HMW-/MMW-adiponectin can modulate dermal fibrotic pathways. However, the current findings were obtained in vitro, and thus do not directly show fibrosis in vivo by HMW-/MMW-adiponectin. In fact, the IHC of skin cGVHD actually showed certain increases in the expressions of fibronectin, TGF-βR2, and MMP-3, but not of TIMPs. These IHC findings suggest that cGVHD could not be explained only by adiponectin, although skin biopsy samples from only one patient were too small to establish a definite conclusion. The association between adiponectin and skin cGVHD scores should be evaluated in future prospective trials. Other possible limitations of our study are that the assessment time of the current fibroblast analysis was different from the actual development of skin cGVHD and that not only long-term steroid administration but also autopsy samples might affect our IHC results. The symptoms of cGVHD are diverse and complicated, beyond just simple skin fibrosis. Therefore, the role of HMW-adiponectin in the network of cGVHD in vivo remains to be elucidated. Further basic investigations are needed to clarify how HMW-/MMW-adiponectin can play a role in ECM regulation and the pathophysiology of sclerotic cGVHD in vivo, and whether the adiponectin-pathway could be a target for the treatment of sclerotic cGVHD. This study was partially supported by The Research Award to Jichi Medical University Graduate Student (H.N.). Author contributions: H.N. designed the study, performed experiments, analyzed data, and wrote the manuscript; K.T-S., R. Yamazaki, M.S, Y.T., K.S., M.K., R. Yamasaki, H.W., Y.I, K.K, T.M, M.A., S. Kimura, M.K., S.O., A.T., J.K., S. Kako, and J.N. collected data and gave their advice about the experimental procedures; Y.S. collected and analyzed pathologic findings; Y.K. designed the study, analyzed data, and wrote the manuscript. No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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