Peroxisome Proliferator–Activated Receptor- Ligands Inhibit TGF- 1–Induced Fibronectin Expression in Glomerular Mesangial Cells

The thiazolidinedione (TZD) class of antidiabetic drugs, which are ligands for peroxisome proliferator–activated receptor (PPAR), has been shown to possess potent anti-inflammatory and antineoplastic actions. Here, we show in mesangial cells that PPARagonists inhibit fibronectin expression by transforming growth factor (TGF)1. TGF1 enhanced fibronectin mRNA expression, and this enhancement was abrogated by pretreatment with pioglitazone. Electrophoretic mobility shift assay identified that pioglitazone inhibited TGF1– induced DNA binding of activator protein-1 (AP-1). Pioglitazone inhibited AP-1 reporter activity but not Smad binding elements reporter activity without affecting TGF1–induced activation of mitogen-activated protein kinases (MAPKs) or Smad2. PPARoverexpression inhibited TGF1–induced fibronectin expression as well as the activation of AP-1. 15-Deoxyprostaglandin J2 (15d-PGJ2), a natural PPARligand, also inhibited TGF1–induced fibronectin expression by suppressing AP-1 activation by TGF1. 15d-PGJ2 inhibited the TGF1–induced MAPK activation. Dominant-negative PPAR( PPAR) completely abrogated the inhibitory effect of pioglitazone and incompletely blocked its effect of 15d-PGJ2 on TGF1–induced AP-1 reporter activity. PPARoverexpression did not affect the inhibitory effect of 15d-PGJ2 on TGF1–induced MAPK activation. In conclusion, pioglitazone inhibits TGF1–induced fibronectin expression by inhibiting AP-1 activation dependent on PPAR, while 15d-PGJ2 acts through a dual mechanism independent of and dependent on PPARactivation in mouse mesangial cells. Diabetes 53:200–208, 2004 Diabetic nephropathy is characterized by renal hypertrophy, glomerular and tubular basement thickening, and mesangial matrix expansion with extracellular matrix (ECM) protein accumulation (1). High glucose itself and various growth factors were shown to contribute to the accumulation of ECM proteins in the kidney during diabetes (2). Among them, transforming growth factor (TGF)has been proposed as a key cytokine that enhances ECM protein synthesis, leading to the development of diabetic nephropathy (2,3). Indeed, studies (4,5) using neutralizing anti– TGFantibodies have provided convincing evidence that inhibiting TGFactivity could prevent and even reverse the pathological and functional abnormalities in the kidney of diabetic animals. TGFexerts its multiple biologic actions by activating several intracellular signal transduction systems. The Smad family of proteins has been recently identified as a predominant signal transducer of TGF(6). Mitogenactivated protein kinases (MAPKs), including the extracellular signal–regulated kinases (ERKs), the c-jun NH2terminal kinases, and the p38MAPK (7), have also been proposed to participate in TGF–induced ECM protein synthesis in a number of different cell types, including mesangial cells (8–13). Thiazolidinedione (TZD) compounds, such as rosiglitazone and pioglitazone (14), a new class of antidiabetic agents, act by increasing insulin sensitivity and are widely used for the treatment of type 2 diabetic subjects. The antidiabetic effect of TZDs was shown to be mediated through peroxisome proliferator–activated receptor (PPAR), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors (15). PPARforms a heterodimer with retinoid X receptor, and the complex binds to a PPAR–responsive element (PPRE) in the promoter of target genes in response to a variety of endogenous and exogenous ligands (16). PPARis also activated by a natural ligand 15-deoxy-prostaglandin J2 (15d-PGJ2) (17,18). PPARis expressed at high levels in adipose tissue (17), colon and activated macrophages, and at lower levels in other tissues (14,19–21). Although the antidiabetic effect of TZDs is well recognized, we previously reported (22) a novel action of these agents in that TZDs prevented not only glomerular dysfunction such as hyperfiltration and albuminuria, but From the Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan. Address correspondence and reprint requests to Masakazu Haneda, MD, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga, Japan. E-mail: [email protected]. Received for publication 3 June 2003 and accepted in revised form 15 October 2003. 15d-PGJ2, 15-deoxy-prostaglandin J2; PPAR; dominant-negative peroxisome proliferator–activated receptor; AP-1, activator protein-1; dCTP, deoxycytidine triphosphate; DMEM, Dulbecco’s Modified Eagle’s Medium; ECM, extracellular matrix; EMSA, electrophoretic mobility shift analysis; ERK, extracellular signal–regulated kinase; FBS, fetal bovine serum; MAPK, mitogen-activated protein kinase; PPAR, peroxisome proliferator–activated receptor; PPRE, PPAR-responsive element; SBE, Smad-binding element; TGF, transforming growth factor; TZD, thiazolidinedione. © 2004 by the American Diabetes Association.