The FASEB Journal express article10.1096/fj.02-1117fje. Published online April 22, 2003.
Advanced glycation end products activate Smad signaling via TGF-β-dependent and -independent mechanisms: implications for diabetic renal and vascular disease Jin H. Li,* Xiao R. Huang,* Hong-Jian Zhu,† Matthew Oldfield,‡ Mark Cooper,‡ Luan D. Truong,* Richard J. Johnson,* and Hui Y. Lan* *Departments of Medicine-Nephrology and Pathology, Baylor College of Medicine, Houston, TX; †Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Melbourne, Australia; ‡ Department of Medicine, Austin and Repatriation Medical Center, The University of Melbourne, Melbourne, Australia Correspondending author: Hui Y. Lan, Department of Medicine-Nephrology, Baylor College of Medicine, One Baylor Plaza, Alkek N520, Houston, TX 77030. E-mail:
[email protected] ABSTRACT While it is thought that advanced glycation end products (AGEs) act by stimulating transforming growth factor (TGF)-β to mediate diabetic injury, we report that AGEs can activate TGF-β signaling, Smads, and mediate diabetic scarring directly and independently of TGF-β. AGEs activate Smad2/3 in renal and vascular cells at 5 min, peaking over 15–30 min before TGF-β synthesis at 24 h and occurs in TGF-β receptor I and II mutant cells. This is mediated by RAGE and ERK/p38 mitogen-activated protein kinases (MAPKs). In addition, AGEs also activate Smads at 24 h via the classic TGF-β-dependent pathway. A substantial inhibition of AGEinduced Smad activation and collagen synthesis by ERK/p38 MAPK inhibitors, but not by TGFβ blockade, suggests that the MAPK-Smad signaling crosstalk pathway is a key mechanism in diabetic scarring. Prevention of AGE-induced Smad activation and collagen synthesis by overexpression of Smad7 indicates that Smad signaling may play a critical role in diabetic complications. This is further supported by the findings that activation of Smad2/3 in human diabetic nephropathy and vasculopathy is associated with local deposition of AGEs and upregulation of RAGE. Thus, AGEs act by activating Smad signaling to mediate diabetic complications via both TGF-β-dependent and -independent pathways, shedding new light on the pathogenesis of diabetic organ injury. Key words: TGF-β/Smad signaling • Smad7 • AGEs and RAGE • diabetic nephropathy and vasculopathy
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here is increasing evidence of a causal role for advanced glycation end products (AGEs) in the development of diabetic complications, including nephropathy and vascular disease (1, 2). AGEs exert effects both directly through the formation of protein cross-links that alter the structure and function of extracellular matrix (ECM) and by interacting with specific cell surface receptors (1–5). The best characterized AGE receptor is RAGE (5), although other AGE binding sites have also been reported (7, 8). Engagement of RAGE by AGEs leads to the
activation of several intracellular signaling pathways, including mitogen-activated protein kinase (MAPK), nuclear transcription factor (NF)-κB, and activating protein (AP)-1 and increases the production of cytokines, including transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF)-α (9–12). Successful prevention and treatment of diabetic complications by blocking AGE and RAGE activities have further indicated the pathogenic importance of AGEs (13–15). This is further confirmed by a recent study in RAGE-overexpressing mice in which the pathophysiological significance of interactions between AGEs and RAGE in the development of diabetic nephropathy has been demonstrated (16). Although the involvement of AGE-RAGE interaction in inflammatory disorders, including diabetes, has been well established (1, 17), the intracellular signaling pathways of AGE-mediated tissue scarring remain unclear. Several studies suggest that AGEs may activate TGF-β to cause renal and vascular sclerosis (12, 18–23). TGF-β has been recently shown to play a central role in the development of diabetic nephropathy (20, 21), and this is further supported by the finding that blockade of TGF-β, using a neutralizing antibody or antisense strategies, attenuates diabetic organ injury in mice (24–26). However, it remains unclear how AGEs signal cells to produce ECM and thereby induce tissue scarring, a common pathological hallmark of diabetes-related organ injury. The recent discovery of the TGF-β/Smad signaling pathway has allowed investigation of intracellular mechanisms of TGF-β in diabetic complications. TGF-β is known to mediate its fibrotic effects by activating the receptor-associated Smads, including Smad2 and Smad3, which is counterregulated by inducing its negative regulating Smads, that is, Smad6 and Smad7 (27– 29). It has been shown that Smad3 is activated in diabetic db/db mice (30). We tested whether the Smad signaling pathway is activated in human diabetic nephropathy and vasculopathy and report here that AGEs activate the Smad signaling pathway directly and independently of TGF-β, in addition to the classic TGF-β-dependent Smad signaling pathway. Importantly, we demonstrated that targeting the activated Smad signaling pathway by overexpression of inhibitory Smad7 was able to block AGE-induced ECM production in vitro. MATERIALS AND METHODS Patients Kidney tissues (10 autopsies and 27 biopsies) with unequivocal diabetic nephropathy were obtained from the Department of Pathology, Baylor College of Medicine. The study was approved by the University Review Board. The renal pathological changes included diffuse glomerulosclerosis (9 cases), nodular glomerulosclerosis (28 cases), and moderate-to-severe glomerulosclerosis and tubulointerstitial fibrosis (31 cases). All patients had moderate-to-severe proteinuria (4.65±0.8 g/day), increased serum creatinine (2.9±0.3 mg/dl), and decreased creatinine clearance (36.0±3.7 ml/min). Establishing doxycycline (Dox)-regulated Smad7-expressing tubular epithelial cell line The Dox-regulated flag-Smad7-expressing normal rat renal tubular epithelial cell line (NRK52E) was established as described previously (31). The stable transfected cells were selected in the presence of puromycin (2 µg/ml). Positive clones were confirmed by their ability to express
Smad7 in the presence of Dox by Western blot analysis, using an anti-flag M2 antibody (IBI, Eastman Kodak, Rochester, NY). The clone S7–7 was used in this study. Cell culture Three cell lines, including normal rat tubular epithelial cells (TECs) (NRK52E), normal rat glomerular mesangial cells (MCs, 1097), and normal rat vascular smooth muscle cells (VSMCs), were used in this study. In addition, two TGF-β receptor (TβR) mutant mink lung epithelial (Mv1Lu) cell lines—R1B cells, which lack functional TβRI, and DR26 cells, which contain a truncated, nonfunctional TβRII (gifts from A. B. Roberts, National Institutes of Health, Bethesda, MD)—were also used (32). To investigate the negative regulating role of Smad7 in AGE-induced activation of Smad 2/3, the stable, Dox-regulated Smad7-expressing NRK52E cells were applied as described previously (31). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, BRL, Gaithersburg, MD) containing 0.5% fetal bovine serum in six-well plastic plates or eight-chamber glass slides (Nunc, Naperville, CT) at 37°C. Cells were stimulated with AGE-BSA or BSA control at concentrations of 0, 10, 33, and 100 µg/ml for periods of 5, 15, 30, and 60 min and 2, 6, 12, and 24 h for Smad2 and 3 detection and for periods of 1, 3, and 5 days for collagen matrix measurement. The preparation and degree of modification of AGE-BSA and control BSA were described previously (22). All AGE-BSA preparations and dilutions were performed under endotoxin-free conditions and were passed over an endotoxin binding affinity polymyxin column (Detoxi-gel, Pierce, Rockford, IL). Before in vitro study, reagents were tested for endotoxin levels by the Limulus ameboecyte assay (EToxate, Sigma, St. Louis, MO) and were found to contain
