Organfibrosis inhibited by blocking transforming growth factor-β signaling via peroxisome proliferator-activated receptor γ agonists

Chengdu,China

Introduction

Organfibrosis has been viewed as one of the major medical problems,which can lead to progressive dysfunction of the liver,lung,kidney,skin,heart,and eventually the death of patients.[1-6]No effective treatment is available at present.Fibrosis is initiated by a variety of pathological,physiological,biochemical and physical factors.Except the different etiologies of the disease,there is a common pathogenetic process:excessive activation of the key profibrotic cytokine,transforming growth factor-β(TGF-β),which increases the synthesis of extracellular matrix (ECM) proteins and decreases the degradation associated with a gradual destruction of normal tissue and function (Fig.1).[2-6]Furthermore,TGF-β signaling plays an important role in the epithelial-tomesenchymal transition (EMT).Although there is a normal development process,EMT can be induced by excessive activation of TGF-β in various epithelial cells after organ injury,with loss of epithelial characteristics and presence of mesenchymal features (Fig.2).[7-9]Thus lots of ECM proteins were synthesized in the liver,lung,kidney and heart.[8-11]The TGF-β signal transduction pathway therefore has become an effective target for the treatment offibrotic diseases.

Many studies have shown that on activation by two important classes of ligands:natural ligand (15d-PGJ2)or synthetic ligand (TZDs),peroxisome proliferatoractivated receptor γ (PPARγ) can block the TGF-β signaling pathway and then suppress tissue and organfibrosis.PPARγ,ubiquitously expressed throughout the body,belongs to ligand-activated transcription factors of the nuclear receptor superfamily.[12,13]Through the formation of a heterodimer with retinoid X receptor α(RXRα),PPARγ subsequently binds to the peroxisome proliferating response element (PPRE),known as a specific DNA sequence in the promoter region of PPARγ target genes,to turn on or turn off the transcription of different genes including glucose and lipid metabolism,in flammation,fibrosis and cancer.[14-16]Here we review the recent advances in understanding how PPARγ agonists inhibit TGF-β-induced profibrotic effects in the liver,lung,kidney,skin,heart and other organs,with particular emphasis on the main antifibrotic mechanism.

Fig.1.The central role of TGF-β infibrosis.A variety of pathology,physiology,biochemistry and physical factors stimulate TGF-β expression,ultimately resulting infibrosis.Ang II:angiotensin II;TX:thromboxane; ROS:reactive oxygen species.

TGF-β and organfibrosis

TGF-β contributes to the pathologicalfibrosis in most organs,especially the liver,kidney,lung and skin,a topic that has been the subject of numerous studies (Table 1).Moreover,excessive TGF-β can induce EMT in various epithelial cells,which also involved in organfibrosis(Table 2).

Liverfibrosis

Liverfibrosis is the common pathological process in chronic liver diseases caused by various factors.[37-39]After liver injury,the activated TGF-β signaling promotes the activation and proliferation of hepatic stellate cells (HSCs) known as the major source of ECM,thereby inducing excessive ECM deposition.[38-42]High levels of TGF-β1 are often found in liverfibrosis,and there may be a significantly positive correlation between the elevated TGF-β1 mRNA level andfibrogenic activity.[17-19]For example,the expression of TGF-β1 mRNA increases 12-fold in HSCs fromfibrotic rat livers over that in normal HSCs.[19]

Fig.2.Epithelial-mesenchymal transition (EMT).

Table 1.Diseases characterized by excessive expression of TGF-β

Table 2.Role of TGF-β-induced EMT in variousfibrotic diseases

Increasing evidence supports that TGF-β1-induced EMT plays a potentially crucial role in liver fibrogenesis,[43-46]resulting in accumulation of activatedfibroblasts associated with significant deposition of ECM.[28,29,42]Conversely,the inhibition of TGF-β1-induced EMT may be a target for further therapeutic intervention in liverfibrosis.The blockade of TGF-β1 signaling with bone morphogenetic protein-7 (BMP-7)significantly eliminates EMT-derivedfibroblasts and hence attenuates liverfibrosis in mice.[29]Similarly,mouse hepatocytes Smad7 expression can improve CCl4-provoked liver damage andfibrosis scores by inhibiting TGF-β1-induced EMT.[47]

Pulmonaryfibrosis

Failure to restore the alveolar epithelial cell properties after injury is an important feature of pulmonaryfibrosis.Therefore,lungfibroblasts are used to replace the damaged cell or tissue and differentiate into myofibroblasts via EMT associated with excessive ECM accumulation in the interstitium,which progressively disrupts lung function and ultimately leads to respiratory failure.[48-51]TGF-β,a key mediator in pulmonaryfibrosis,has received much attention.[52-54]Significantly high levels of TGF-β1 are detected in patients with pulmonary disease andfibrosis.[20,21]It was reported that levels of TGF-β1 and TGF-β3 mRNAs were significantly higher in lung biopsies from Iranian victims of chemical gases.[20]And lung biopsies from patients with idiopathic pulmonaryfibrosis showed that the mean TGF-β1 levels were also significantly un-regulated.[21]

In addition,TGF-β induces the activation and proliferation of lungfibroblasts by EMT.[30-32]It is well known that alpha-smooth muscle actin (α-SMA) is a key marker of myofibroblast differentiation.And in vitro,this protein can be significantly induced through the activation of TGF-β1/Smad3 signaling pathways.[31,32]On the other hand,TGF-β induces EMT in alveolar epithelial cells (AECs) in vitro and in vivo,[30-32]making AECs a source of myofibroblasts,promoting the expression of ECM proteins and connective tissue growth factor(CTGF).[33,34]

Renalfibrosis

Renalfibrosis,characterized by an abnormal deposition of ECM,is the common end-stage of most progressive kidney diseases.[55,56]This pathology usually involves in the activation of multiple signaling molecules,such as TGF-β and angiotensin II.[55-57]Among them,TGF-β is considered as the most powerful profibrotic cytokine.[58-61]TGF-β1 level is positively correlated with the grade of renal tubular injury and renal interstitialfibrosis in IgA nephropathy.[22]Caletti et al[24]found that TGF-β1 excretion is significantly higher in patients with D-plus hemolytic uremic syndrome than in the healthy controls.In vivo,transgenic mice overexpressing TGF-β1 in renal tubules significantly contribute to renal tubulefibrosis.[25]

TGF-β1-induced EMT can also occur in tubular epithelial cells,which induce myofibroblast differentiation and accumulation followed by the excessive ECM deposition in the interstitium,leading to progressive renalfibrosis and ultimately renal failure.Hence,inhibition of TGF-β1-induced EMT may be a novel route for attenuating renalfibrosis.[35,36,62,63]For example,lefty or BMP-7,known to antagonize the actions of TGF-β1,has been shown to attenuate renal dysfunction by reversing the TGF-β1-induced EMT.[35,36]

Keloids

Keloids formation is a proliferative response to dermal injury such as trauma,surgery,burns and in flammation,leading to continuous synthesis of collagen in the dermis and subcutaneous tissues.[64,65]The precise pathogenesis of keloid formation is unknown,but presumably TGF-β is a key factor in keloids formation after injury,which induces the migration of mesenchymal cells into the wound and stimulates their proliferation,thereby resulting in excessive ECM synthesis.[66,67]Keloidfibroblasts are extremely sensitive to TGF-β.[68,69]Elevated levels of TGF-β1 and its receptors have often been found in keloidfibroblasts.[26,27]Treatment with TGF-β1 results in the overproduction of collagen in keloidfibroblasts.Conversely,intervention of TGF-β1 signal shows a potential therapeutic effect for keloid.[70-72]

Inhibition of TGF-β-induced organfibrosis by PPARγ agonists

PPARγ-mediated regulation of gene expression can be divided into two classes:direct regulation and indirect regulation.The former is the classical and main regulation,in which activation of PPARγ binds to a PPRE sequence,directly inducing target gene transcription.Indirect regulation,which does not require DNA binding by PPARγ.PPARγ directly interacts with another protein,or competes with other nuclear receptors for a common RXRα partner to prevent some regulators such as AP-1,NF-κβ,TGF-β or STAT from binding to their own response elements,regulating the gene expression.[73]In addition,the high expression of PPARγ has been found throughout the body.Thus,the activation of PPARγ may show antifibrotic properties in many organs,which has been confirmed by numerous studies.And these studies found that the main antifibrotic effect is blockade offibrogenic TGF-β signaling by PPARγ agonists (Table 3,Fig.3).

Liver

The activation and proliferation of HSCs are the central event in liver fibrosis.In activated HSCs,there is an antagonistic relationship between PPARγ activation and TGF-β signaling.Activation of PPARγ suppresses the TGF-β signaling pathway,whereas exogenous TGF-β1 can inhibit the expression of PPARγ.[91]In vitro,exogenous TGF-β1 induces excessive ECM proteins accumulation in human adipocytic HSCs by a timeand dose-dependent up-regulation of plasminogen activator inhibitor type I (PAI-I) expression,which is eliminated by decreasing transcription of the PAI-I gene promoter by PPARγ agonist GW7845 without any cytotoxicity.[74]It is known that CTGF is an important downstream mediator of TGF-β-inducedfibrosis,[92]and overexpression of CTGF greatly facilitates the process of tissue and organfibrosis.In cultured rat HSCs,PPARγ activated by 15d-PGJ2 or GW7845 binds to a sequence of the CTGF promoter,dramatically suppressing TGF-β1-induced CTGF expression at both the mRNA and protein levels.But pretreatment with the PPARγ antagonist GW9662 shows the reversed inhibitory effect,further suggesting that the inhibition is indeed mediated by PPARγ.[74]

Fig.3.Regulation of TGF-β-induced profibrotic effects by PPARγ.The activation of PPARγ by agonists disrupts the TGF-β/Smad signal transduction,showing antifibrotic properties in many organs and tissues.The PPARγ antagonist GW9662 abrogates this effect.

Lung

The differentiation and persistence of myofibroblasts in the lung are considered to be important in the development and progression of pulmonaryfibrosis.Fibroblasts can differentiate into myofibroblasts in vitro on exposure to TGF-β.When exposed to PPARγ agonists such as 15d-PGJ2,ciglitazone and rosiglitazone,TGF-β1-driven myofibroblast differentiation is significantly inhibited and followed by reduction of collagen production.[32,76]Similarly,the novel PPARγ ligand,2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid attenuate the up-regulation of α-SMA,FN and collagen by inhibition of the differentiation of TGF-β1-stimulated myofibroblasts.[77]Interestingly,Lin et al[78]reported that rosiglitazone treatment can reduce the migration distance of lungfibroblasts stimulated by fetal bovine serum and TGF-β1 in vitro.Moreover,the effects of PPARγ agonists are not limited tofibroblasts; they also inhibit TGF-β1-induced EMT in AECs.Rosiglitazone and ciglitazone inhibit the reduction of E-cadherin and the elevation of N-cadherin and collagen I induced by TGF-β1 in A549 cells,but show no effects on elongatedfibroblast-like cells.[79]To identify the potential interactions of PPARγ and TGF-β in pulmonaryfibroblasts and AECs will provide further understanding of the antifibrotic properties of PPARγ agonists.

Kidney

PPARγ agonists also exert potentially antifibrotic actions in renalfibrosis by blocking TGF-β signal transduction.In vitro exogenous TGF-β1 stimulation promotes the mRNA and protein expression of ECM components in both human and mouse glomerular mesangial cells (particularly FN),whereas pioglitazone reverses this effect.[80,81]It has been reported that the cyclic AMP-responsive element binding protein (CREB)and the TGF-β/Smads signaling pathway cooperate to mediate TGF-β1-induced glomerulosclerosis,suggesting a tight interaction between the TGF-β/Smads and the PKA/CREB signal pathway,[93]which is important for the glomerulosclerosis.The PPARγ agonist troglitazone or telmisartan efficiently suppresses TGF-β/PKA signaling followed by the inhibition of rat mesangial cells activation and ECM synthesis.And the inhibitory effect can be abolished by pre-incubation with GW9662.[82]Besides mesangial cells,a similar phenomenon has also been reported in rat renal interstitialfibroblasts(NRK/49F) in vitro.[83]More direct evidence is that the administration of exogenous TZDs attenuates the obstructed kidneyfibrosis in C57BL/6J mice through the reduction of TGF-β expression.[84]

In addition,the relationship between Angiotensin II and TGF-β in the kidney has also aroused much interest.[94]Angiotensin II increases TGF-β mRNA expression in cultured mesangial cells,whereas blocking TGF-β signaling eliminates the stimulatory effect of Angiotensin II on matrix protein synthesis.[95,96]In view of these results,we speculate that PPARγ agonists reduce kidneyfibrosis by suppressing the Angiotensin II-TGF-β axis.Recently,klotho,a single-pass transmembrane protein expressed in renal tubular epithelial cells,whose soluble form has been found to directly bind to TβR II and inhibit TGF-β1 signaling,thereby suppressing TGF-β1-induced EMT and renalfibrosis.[97]Interestingly,PPARγ agonists augment klotho expression in the kidney through two upstream non-canonical PPARγ binding sites in the klotho gene,[98,99]suggesting that PPARγ agonists can also indirectly inhibit TGF-β-induced renalfibrosis by increasing the activity of klotho.

Heart

As well as in the kidney,there is a causal relationship between Angiotensin II and TGF-β1 in cardiacfibrosis.[100]In wild-type mice,absence of the TGF-β1 gene fully prevents the development of cardiac hypertrophy induced by excessive Angiotensin II.[101]Furthermore,Angiotensin II can stimulate TGF-β1 mRNA expression by subsequent activation of downstream signaling molecules NAD(P)H oxidase,protein kinase C,p38-MAP kinase,and nuclear AP-1 in cardiomyocytes,ultimately leading to myocyte hypertrophy and cardiacfibrosis (Fig.4),[102]indicating the importance of Angiotensin II-TGF-β axis in cardiacfibrosis.Without doubt,PPARγ is also functionally expressed in the heart.And it has been reported[103]that PPARγ agonists rosiglitazone and 15d-PGJ2 attenuate cardiacfibrosis through interrupting Angiotensin II-induced TGF-β1 expression.

Fig.4.Role of Ang II-TGF-β axis in cardiac fibrosis.AT1R:Ang II type 1 receptor; AP-1:nuclear activating protein-1; βAR:β-adrenergic receptor.

Keloids

As mentioned above,TGF-β1 is a key profibrotic mediator in Keloid formation.It is,therefore,important to identify the mechanism that represses the TGF-β1 signaling pathway.One such mechanism is the induction of Smad2/Smad3,an endogenous negative feedback to terminate TGF-β1 responses.[27,104,105]And in both normal and keloidfibroblasts,PPARγ agonists can repress TGF-β1 responses by inducing the expression of Smad2/Smad3,subsequently limiting TGF-β1 signaling transduction.However,the TGF-β1/Smad2/3 signaling pathway may partly contribute to the inhibition of ECM by PPARγ agonists.[85,86]Other mechanisms may also be involved.For example,TGF-β1 can mediate the activation of mitogen-activated protein kinase,which has been shown to be important in regulating collagen metabolism.[80]

Furthermore,skin and lung biopsies from patients with systemic sclerosis show markedly diminished levels of PPARγ,associated with aberrant repair in these tissues.[106]And excessive TGF-β activity in systemic sclerosis impairs PPARγ function,which triggers afibrotic response,indicating that enhanced sensitivity to exogenous PPARγ agonists may represent a novel treatment strategy for dermalfibrosis,such as hypertrophic scar,keloid,and systemic sclerosis.

PPARγ-independent activities of PPARγ agonists

Whether the effects of PPARγ agonists are really mediated by activating the transcription factor needs careful evaluation,because PPARγ has at least three possible ways to regulate gene expression.[73]While many of the studies in the liver,kidney,lung and skin cited above are descriptive,the similarity of findings suggests that the antifibrotic activity of PPARγ agonists by suppressing the TGF-β signaling pathway is in fact PPARγ-dependent.However,apart from these PPARγ-dependent effects,many effects of PPARγ agonists are not related to PPARγ activation (PPARγ-independent).[80,83]Fox example,15d-PGJ2 inhibitsfibrosis-related gene expression not only by interference with the TGF-β/Smads signaling pathway,but also by directly suppressing the activation of AP-1 and NF-κβ transcription factors,[107,108]suggesting that the inhibitory effects of 15d-PGJ2 are mediated by both PPARγ-dependent and independent mechanisms.

Conclusion and perspectives

It is not surprising that PPARγ agonists exhibit a wide range of antifibrotic effects by inhibiting the TGF-β signaling pathway because of the widespread tissue distribution and complicated functions of PPARγ.Besides the major organs described above,PPARγ agonists also potentially exert antifibrotic activity in other organs and tissues by blocking the TGF-β signaling pathway,such as the pancreas,[87]peritoneal cavity,[88]nose[89]and aorta.[90]In summary,overwhelming evidence has demonstrated that TGF-β is a key mediator offibrotic tissues,and overexpression of TGF-β correlates significantly withfibrogenic activity in various organs and tissues.PPARγ agonists markedly inhibit TGF-β signal transduction and are effective antifibrogenic agents.

Although there are huge advances in PPARγ study,further investigations are needed on action of PPARγ and to know how PPARγ participates in this antifibrotic mechanism since limitations exist in the current clinical use of PPARγ agonists.Especially TZDs,synthetic PPARγ agonists,may actually dominate our decisions on the clinical use of PPARγ agonists because of a widely used therapy for type 2 diabetes.However,TZDs act primarily as effective insulin sensitizers,and inevitably cause side-effects including weight gain,fluid retention,congestive heart failure,and bone fractures.Since such side-effects have become more evident,the use of these drugs has become controversial.[109]In addition,TGF-β1 not only acts as the key profibrotic cytokine,but also plays many essential roles in multiple developmental processes,such as immune regulation,cell differentiation and wound healing,and TGF-β1/Smads signaling forms cross-talk networks with other signal pathways,indicating that blockade of TGF-β by PPARγ agonists certainly affects these cytokine networks and causes unwanted side-effects.In view of these facts,anti-TGF-β therapies with PPARγ agonists may have to be carefully tailored to be tissue- and target gene-specific to minimize side-effects,which is a great challenge to the medical research at present.

Contributors:DYL and CNS contributed to study concept and design,DYL and XXZ to data acquisition,and DYL to drafting of the paper.DYL,XXZ and CNS made critical revision of the paper.CNS and XXZ were the supervisors of the study.

Funding:None.

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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