The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy
Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown.
Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR.
Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ.
These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel pharmacological approach to controlling fibrosis.
Scleroderma or systemic sclerosis (SSc) is a chronic autoimmune disease associated with fibrosis in multiple organs . Fibrosis in the skin is due to overproduction of collagen and other extracellular matrix (ECM) components by activated fibroblasts accompanied by progressive loss of subcutaneous adipose tissue . Transforming growth factor-β (TGF-β) is a key mediator of fibrosis that initiates and sustains fibroblast activation and myofibroblast differentiation . A variety of cell-autonomous regulatory mechanisms exist to control fibroblast activation and prevent aberrant constitutive fibrogenesis. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a pleiotropic nuclear receptor implicated in the regulation of adipogenesis . Emerging evidence also implicates PPAR-γ in ECM accumulation and connective tissue homeostasis, and natural and synthetic PPAR-γ ligands are potent inhibitors of fibrotic responses .
Adiponectin is a multi-functional 30 kD adipokine that regulates insulin sensitivity, energy balance and cellular metabolism . The expression of adiponectin is tightly regulated by PPAR-γ, and its levels in circulation are decreased in patients with obesity, type 2 diabetes and metabolic syndrome . In contrast, serum levels are raised by PPAR-γ agonist treatment in mice and in humans . Significantly, recent studies demonstrate that adiponectin levels are reduced in patients with diffuse cutaneous scleroderma, and are inversely correlated with disease activity, severity and duration [9–12]. These observations point to a potential role for adiponectin in the pathogenesis of scleroderma, but the underlying mechanisms are not currently understood.
Fibrosis in scleroderma is associated with impaired PPAR-γ expression and activity and reduced adiponectin levels, which may be a direct consequence of the PPAR-γ defect [11, 12, 24, 25]. In light of these intriguing recent observations, we sought to gain a better understanding of the role of adiponectin in the modulation of collagen synthesis and myofibroblast differentiation in fibroblasts. Results using two-dimensional monolayer cultures and three-dimensional full-thickness human skin equivalents demonstrate that adiponectin potently suppressed the expression of Type I collagen and α-smooth muscle actin (α-SMA) in normal and scleroderma fibroblasts, and abrogated the stimulation of these responses elicited by TGF-β. The inhibitory effects of adiponectin were mediated by activation of AMP kinase. Moreover, genetic deletion of adiponectin in mouse fibroblasts abrogated the inhibition of TGF-β signaling elicited by PPAR-γ agonists. The expression of adiponectin receptor 1 was selectively reduced in skin biopsies from patients with scleroderma. Taken together, these findings indicate that the adiponectin/AMP kinase pathway may play a previously unrecognized important homeostatic role in ECM regulation, and its defective function contributes to aberrant fibroblast activation in the pathogenesis of fibrosis. The adiponectin signaling pathway, therefore, represents a novel therapeutic target in scleroderma.
Materials and methods
Cell culture and reagents
Primary fibroblast cultures were established by explantation from neonatal foreskin biopsies, or from skin biopsies from healthy adults and scleroderma patients obtained under the protocols approved by the Institutional Review Board at Northwestern University. All donors or their parents/legal guardians provided written informed consent. Mouse skin fibroblasts were established by explant culture from three-week-old adiponectin-null mice and wild-type littermates . Fibroblasts were maintained in (D)MEM) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland), 50 μg/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C, and studied between passages 2 to 8 . When fibroblasts reached confluence, growth media with 10% FBS or serum-free media supplemented with 0.1% BSA were added to the cultures for 24 hours prior to TGF-β2 (Peprotech, Rocky Hill, NJ, USA), or full-length adiponectin (Bio Vendor, Karasek, Czech Republic). In selected experiments, the AMP-activated protein kinase (AMPK) inhibitor Compound C (Sigma, St Louis, MO, USA) was added to the culture 60 minutes prior to adiponectin. Toxicity was determined using lactate dehydrogenase (LDH) assays according to the manufacturer's instructions (Biovision, Milpitas, CA, USA).
Three-dimensional full-thickness human skin equivalents
Normal skin fibroblasts (3 × 105) were suspended in 1.5 ml reconstitution buffer and (D)MEM. Cells were mixed with rat tail type I collagen (4 mg/ml, BD Biosciences, San Jose, CA, USA) and seeded in 12-well plates at 37°C for 48 hours to solidify the collagen plug. Epidermal keratinocytes (6 × 106) were isolated from foreskin and suspended in E medium supplemented with 5 ng/ml epidermal growth factor (EGF) and seeded on the collagen plug [28, 29]. Forty eight hours later, organotypic cultures were placed on a metal grid (BD Biosciences) and maintained at an air-medium interface by feeding with E medium every other day for five days. Metformin (1 mM) was added to the media for 24 hours followed by TGF-β (5 ng/ml). Following incubation for a further six days, cultures were harvested, RNA was isolated, and tissues were fixed in formalin. Paraffin-embedded sections (4 μm thickness) were examined by Picrosirius Red staining.
Short interfering RNA-mediated knockdown and adenovirus infection
Fibroblasts were transfected with target-specific siRNA (Dharmacon, Lafayette, CO, USA) or scrambled control siRNA (10 nM). Twenty-four hours following transfection, fresh media were added to the cultures, and the incubations were continued for a further 24 hours. Knockdown efficiency was evaluated by determining endogenous mRNA levels by real-time qPCR.
RNA isolation and real-time quantitative PCR (qPCR)
Primers used for real-time qPCR.
olg1, 5'-CCCCATGAACGAGGGAATT-3' olg2, 5'-GGGACTTAATCAACGCAAGCTT-3'
olg3, 5'-CATGAGAAGTATGACAACAGCCT-3' olg4, 5'-AGTCCTTCCACGATACCAAAGT-3'
olg20, 5'-CAGGGCTGTTTTCCCATCCAT-3' olg21, 5'-GCCATGTTCTATCGGGTACTTC-3'
olg149, 5'-GCTGGTGTGATGGGATTC-3' olg150, 5'-GGGAACACCTCGCTCT-3'
olg396, 5'-TGACTGGCTAAAGGACAACG-3' olg397, 5'-AAAAGAGAAACAGCACGAAACC-3'
olg400, 5'-CAGCCATTATAGTCTCCCAGTG-3' olg401, 5'-CCGAGATGACATAGTGCAAGG-3'
olg404, 5'-ACTGGACGAGCAACTTCATC-3' olg405, 5'-GAGGACCGCAAATAGAAGGAA-3'
Microarray procedures and data analysis
Expression of AdipoR1/2 mRNA was interrogated in publicly available genome-wide expression scleroderma skin microarray datasets (GEO accession number: GSE9285) .
Transient transfection assays
Fibroblasts at early confluence were transfected with [SBE]4-luc plasmids harboring four copies of a minimal Smad-binding element using SuperFect Transfection kit (Qiagen) as described . Cultures were incubated in serum-free media containing 0.1% BSA for 24 hours, followed by TGF-β2 for a further 24 hours and harvested. Whole cell lysates were assayed for their luciferase activities using a dual-luciferase reporter assay system (Promega, Madison, WI, USA). In each experiment, Renilla luciferase pRL-TK (Promega) was cotransfected as control for transfection efficiency . Transient transfection experiments were performed in triplicate and repeated at least twice with consistent results.
Confocal immunofluorescence microscopy
Fibroblasts (1 × 104 cells/well) were seeded onto eight-well Lab-Tek II chamber glass slides (Nalge Nunc International, Naperville, IL, USA) and incubated in serum-free Eagle's minimal essential medium (EMEM) with 0.1% BSA for 24 hours. Fresh media with adiponectin (5 ug/ml) were added, and the incubations continued for a further 24 hours. At the end of the experiments, cells were fixed, permeabilized, and incubated with primary antibodies to Type I collagen at 1:500 dilution (Southern Biotech, Birmingham, AL, USA), or to α-SMA at 1:200 dilution (Sigma, St Louis, MO, USA). Cells were then washed with PBS and incubated with secondary antibodies at 1:500 dilution (Alexa Fluor 488 and 594, Invitrogen) and viewed under a Nikon C1Si confocal microscope.
At the end of each experiment, fibroblasts were harvested and whole cell lysates subjected to Western analysis as described . The following antibodies were used: Type I collagen (Southern Biotech), α-SMA (Sigma), and GAPDH (Zymed, San Francisco, CA, USA). Bands were visualized using ECL reagents (Pierce, Rockford, IL, USA).
Statistical analysis was performed on Excel (Microsoft, Redmond, WA, USA) using Student t-test or analysis of variance (ANOVA). The results are shown as the means ± SEM. P <0.05 was considered statistically significant.
Adiponectin inhibits collagen and alpha-smooth muscle actin gene expression
Clinical features of SSc patients.
Adiponectin attenuates TGF-β-induced profibrotic responses
Agonists of AMP kinase inhibit fibrotic gene expression and abrogate TGF-β responses
Adiponectin mediates the anti-fibrotic effects of PPAR-γ ligands
Adiponectin attenuates LPS-induced profibrotic responses
Adiponectin receptor expression in scleroderma
To evaluate AdipoR1/2 mRNA expression in scleroderma skin, the expression of these genes was interrogated in a publicly available microarray dataset examining gene expression in skin . Biopsies clustering within the diffuse and inflammatory intrinsic subsets  showed an approximately 30% reduction in AdipoR1 (P <0.05), with a slight reduction in AdipoR2 (P = ns) expression compared to biopsies clustering with the normal-like subset (Figure 8B).
Persistence of activated myofibroblasts in response to chronic TGF-ß signaling underlies the progression of fibrosis in scleroderma . We have demonstrated that PPAR-γ activation by endogenous ligands or pharmacological agonists exerts potent inhibitory effects on collagen gene expression and myofibroblast differentiation, and blocks TGF-ß-induced profibrotic responses, in mesenchymal cells in vitro [37, 38]. Moreover, the PPAR-γ ligand rosiglitazone was shown to prevent and attenuate the development of dermal fibrosis in mice . Significantly, recent studies have revealed a marked impairment of PPAR-γ expression and activity in skin biopsies from subsets of patients with scleroderma . Moreover, explanted scleroderma fibroblasts showed reduced PPAR-γ . We have previously identified a scleroderma subset with impaired PPAR-γ signaling that was associated with a strong 'TGF-ß-activated gene signature' in skin biopsies . These scleroderma patients had a rather aggressive form of disease with extensive skin fibrosis. While these findings strongly implicate aberrant PPAR-γ function in the persistent fibrosis of scleroderma, the underlying molecular mechanisms remain to be elucidated.
The present studies showed that the PPAR-γ-regulated adipokine adiponectin caused a marked inhibition of collagen gene expression and myofibroblast differentiation in neonatal and normal adult skin fibroblasts as well as in scleroderma fibroblasts. Significantly, these inhibitory effects occurred at adiponectin concentrations approximating physiological plasma levels (5 to 20 μg/ml) [11, 43]. Adiponectin stimulated the expression of BAMBI, an endogenous negative regulator of Smad-dependent signaling, while blocking fibrotic responses elicited by TGF-β, as well as by the TLR4 ligand LPS. While TGF-β-induced collagen production and myofibroblast transformation are known to be mediated via the canonical Smad signaling pathway , the mechanism underlying the fibrotic responses elicited by TLR4 ligands remain incompletely understood. A comparable antagonism between adiponectin and LPS was described in the context of LPS-dependent fibrogenesis in adventitial fibroblasts . The inhibitory effects of adiponectin on fibrotic responses were associated with activation of AMP kinase, a stress-induced metabolic master switch that plays a key role in maintaining energy homeostasis. By detecting and responding to cellular nutrient and energy fluctuations, heterotrimeric AMP kinase promotes catabolic energy-producing pathways to enhance cellular glucose uptake, fatty acid oxidation, and GLUT4 biogenesis . In the present studies, pharmacological AMP kinase agonists mimicked the inhibitory effect of adiponectin on profibrotic gene expression and Smad-dependent signaling, while the selective AMP kinase inhibitor Compound C rescued TGF-β stimulation of fibrotic genes in the presence of adiponectin. Moreover, transient transfection experiments indicate that AMP kinase attenuation resulted in abrogation of canonical Smad-dependent TGF-β signaling. While previous studies have highlighted the anti-inflammatory, anti-oxidant and fatty acid-regulating activities of AMP kinase [18, 47], the present studies reveal important functions for adiponectin in modulating fibrogenesis. The mechanism underlying the anti-fibrotic activities of adiponectin and their significance in health and fibrosis remains to be elucidated.
Adiponectin is an adipocyte-derive pleiotropic hormone with key protective roles in diabetes and atherosclerosis [17, 48, 49]. Sequence-specific recognition of the adiponectin gene promoter PPRE element by activated PPAR-γ results in enhanced adiponectin transcription . Recent studies expand the spectrum of the biological activities ascribed to adiponectin, including important roles in regulating inflammation and cancer . Cellular adiponectin responses are mediated via the seven transmembrane domain type 1 and type 2 adiponectin receptors as well as T-cadherin . Obesity is associated with reduced expression of adiponectin receptors in various tissues, contributing to a state of adiponectin resistance .
We and others have shown that adiponectin levels are reduced in the serum and lesional skin from patients with scleroderma [10–12]. Adiponectin levels were inversely correlated with the skin score, a measure of fibrotic skin involvement, and scleroderma patients with the most extensive skin fibrosis had the lowest adiponectin levels . Moreover, patients responding to anti-fibrotic treatment with improved skin scores or lung function displayed a time-dependent increase in serum adiponectin levels [11, 12].
The inverse correlation between adiponectin signaling and fibrogenesis in scleroderma in the aforementioned studies suggests a potential role for adiponectin in the pathogenesis of skin fibrosis. We are struck by the parallels between reduced adiponectin and disappearance of fat tissue in liver fibrosis on the one hand, where quiescent fat-strong hepatic stellate cells transition into fibrogenic myofibroblasts with down-regulation of PPAR-γ, and loss of subcutaneous adipose tissue associated with dermal fibrosis in patients with scleroderma. These parallels raise the intriguing possibility that subcutaneous adipocytes fulfill a role for analogues to that of the hepatic stellate cells of the skin.
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