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Advanced glycation end products activate Smad signaling via TGF-ß-dependent and independent mechanisms: implications for diabetic renal and vascular disease¹

JIN H. LI, XIAO R. HUANG, HONG-JIAN ZHU^*, MATTHEW OLDFIELD, MARK COOPER, LUAN D. TRUONG, RICHARD J. JOHNSON and HUI Y. LAN²

Departments of Medicine-Nephrology and Pathology, Baylor College of Medicine, Houston, Texas, USA;
^* Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Melbourne, Australia; and
Department of Medicine, Austin and Repatriation Medical Center, The University of Melbourne, Melbourne, Australia

²Correspondence: Department of Medicine-Nephrology, Baylor College of Medicine, One Baylor Plaza, Alkek N520, Houston, TX 77030, USA. E-mail: hlan{at}bcm.tmc.edu

SPECIFIC AIMS

Advanced glycation end products (AGEs) play a critical role in diabetic nephropathy and vasculopathy, but the intracellular signaling pathways remain poorly understood. The present study investigated the role of Smad signaling pathways in AGE-mediated diabetic renal and vascular complications.

PRINCIPAL FINDINGS

1. Smad signaling is activated in human diabetic nephropathy and vasculopathy and is associated with AGE deposition and receptor for AGE (RAGE) up-regulation
We first examined whether Smad signaling is activated in human diabetic nephropathy and vasculopathy and the possible mechanism involved. In contrast to the normal human kidney, strong AGE deposition, RAGE up-regulation, and marked activation of Smad2 and Smad3 (as identified by their nucleated location) were colocalized in diabetic lesions, accounting for up to a 5-fold increase in diabetic glomerulopathy, a 20-fold increase in fibrotic tubulointerstitium, and an 8-fold increase in vasculopathy (see online submission). These findings suggest that AGE-RAGE interaction is associated with activation of Smad signaling, resulting in diabetic nephropathy and vascular complications.

2. AGEs induce rapid Smad activation directly and independent of TGF-ß
A new and significant finding in this study is that AGEs activate Smad signaling to mediate renal and vascular sclerosis directly and independent of TGF-ß. This is supported by the findings: 1) AGEs induce rapid Smad2 and Smad3 nuclear translocation and phosphorylation in normal rat glomerular mesangial cells (MCs), TECs, and smooth muscle cells (VSMCs) as early as 5 min, peaking over 15–30 min, and declining to baseline levels at 2 h (Fig. 1 a); 2) this early activation of Smads occurs in the absence of TGF-ß since there was no TGF-ß detectable within the medium by ELISA and because AGE-stimulated TGF-ß expression was first found at 24 h; 3) AGEs, but not TGF-ß, are able to activate Smads at 5 min, peaking over 15–30 min in TGF-ß receptor I (TßRI, R1B) and TßRII (DR26) mutant cells (Fig. 1b ).

View larger version (86K): [in this window] [in a new window]   Figure 1. AGEs activate Smad signaling via the MAP kinase-dependent and TGF-ß-dependent pathways as demonstrated by Western blots (for Smad2) and immunoprecipitation (for Smad3). a) AGEs induce rapid Smad2 and Smad3 phosphorylation by MCs and VSMC in a time- (33 µg/mL) and dose- (30 min) dependent manner. b) AGEs (33 µg/mL), but not TGF-ß (10 ng/mL), induce Smad activation in TßRI (R1B) and TßRII (DR26) defective cells. c) AGEs induce Smad2 phosphorylation again at 24 h. d) Activation of Smad 2 by AGEs in VSMCs at both peaks (30 min and 24 h) is blocked by a neutralizing RAGE antibody (10 µg/mL) and by ERK1/2 (PD98059, 10 µM) and p38 MAP kinase (SB203580, 10 µM) inhibitors, whereas AGE-induced Smad2 phosphorylation at 24 h is blocked by an anti-TGF-ß antibody (10 µg/mL) but not by a normal rabbit IgG (NRb, 10 µg/mL). Data represent 4 independent experiments.

3. AGEs rapidly activate Smad signaling via the RAGE-mediated, ERK/p38 MAP kinase-dependent cross-talk pathway
We next explored the intracellular mechanism by which AGEs activate Smad signaling directly and independent of TGF-ß. AGEs signal through RAGE to induce rapid Smad activation via the ERK/p38 MAP kinase-dependent mechanism. This is supported by the findings: 1) AGE-induced phosphorylation of ERK/p38 MAP kinases and Smad2/3 is paralleled with the first peak at 15–30 min, which was blocked by a neutralizing anti-RAGE antibody (see online submission); and 2) this early activation of the Smad signaling pathway by AGEs was also blocked by specific MAP kinase inhibitors to ERK1/2 (PD98059) and p38 (SB203580) (Fig. 1d ). To confirm AGE-activated Smad signaling directly via the MAP kinase–Smad signaling pathway and independent of TGF-ß, ERK and p38 MAP kinase inhibitors were added to AGE-stimulated TßRI and TßRII mutant cells. Similarly, AGE-induced Smad2 nuclear location and phosphorylation at 15 min were completely inhibited by the addition of PD98059 or SB203580 (see online submission).

4. AGEs signal through RAGE to activate Smad signaling at 24 h via the classic TGF-ß signaling pathway
AGEs induced a second peak of activation of Smad2 and Smad3 at 24 h (Fig. 1c ). In contrast to the early activation of Smad2/3 at 15–30 min, Smad activation at 24 h was TGF-ß dependent since it was associated with a eightfold increase in TGF-ß in the medium (P<0.01) and the neutralizing TGF-ß antibody inhibited the second peak of AGE-induced Smad2 phosphorylation (Fig. 1d ). Further studies demonstrated that AGEs signaled through RAGE to induce TGF-ß synthesis over 12–24 h via the ERK/p38 MAP kinase mechanism since a neutralizing RAGE antibody and inhibitors to ERK/1/2 and p38 MAP kinases blocked TGF-ß production by AGEs (see online submission) and inhibited Smad2 phosphorylation at 24 h (Fig. 1d ).

5. Blockade of the ERK/p-38 MAP kinase-Smad signaling cross-talk pathway inhibits AGE-stimulated collagen matrix production by renal and vascular cells
The functional importance of AGE-induced ERK/p-38 MAP kinase and TGF-ß-dependent Smad signaling in collagen expression was investigated. Results showed that AGEs were able to significantly up-regulate type I collagen mRNA and protein expression by TECs, MCs, and VSMCs at day 1 with further increase at day 3, which was blocked by ERK (PD98059) and p38 (SB230580) MAPK inhibitors. By contrast, neutralizing TGF-ß antibody caused only partial inhibition of AGE-induced collagen I mRNA and protein expression on day 3 (see online submission). This suggests that the initial RAGE-mediated, MAP kinase-Smad signaling may be a major pathway for AGE-induced collagen matrix production in diabetic complications.

6. Overexpression of Smad7 blocks AGE-induced Smad activation and collagen matrix production
A critical role for Smad signaling in diabetic renal and vascular sclerosis was further demonstrated by inducing overexpression of the inhibitory Smad7 in a stable, doxycycline (Dox) -regulated Smad7 expressing TEC line (NRK52E) in which Smad7 expression is tightly controlled by the concentrations of Dox. Strikingly, Dox-induced overexpression of Smad7 resulted in complete prevention of AGE-induced Smad2 nuclear translocation and phosphorylation (Fig. 2 a) and collagen types I and IV mRNA and protein expression (Fig. 2b ), indicating that Smad signaling is a central pathway for the development of diabetic complications.

View larger version (89K): [in this window] [in a new window]   Figure 2. Overexpression of Smad7 inhibits AGE-induced Smad2 activation and collagen matrix synthesis in vitro. a) Immunocytochemistry and Western blot analysis demonstrate that Dox (2 µg/mL) -induced overexpression of Smad7 transgene (m2Smad7) abolishes AGE (33 µg/mL) -induced Smad2 transnuclear location and phosphorylation at both peaks (15 min and 24 h). b) Dox (2 µg/mL) -induced Smad7 overexpression inhibits collagens I and IV mRNA (by RT-PCR) and protein expression (Western). Data represent 4 independent experiments.

CONCLUSIONS AND SIGNIFICANCE

Smad signaling is activated in human diabetic nephropathy and vascular disease. As shown in Fig. 3 , AGEs activate the Smad signaling pathway and mediate renal and vascular fibrosis through TGF-ß-dependent and independent mechanisms. Substantial inhibition of AGE-induced Smad activation and collagen production by ERK/p38 MAP kinase inhibitors, to a lesser extent by an anti-TGF-ß antibody, indicates that the MAP kinase-Smad signaling cross-talk pathway is a key mechanism in the pathogenesis of AGE-mediated diabetic scarring. Blockade of the AGE-mediated TGF-ß/Smad signaling pathway and collagen production by overexpression of the inhibitory Smad7 indicates that Smad signaling is a central pathway of diabetic renal and vascular scarring. These data imply that diabetic renal and vascular sclerosis is regulated by TGF-ß/Smad signaling and that targeting the Smad signaling pathway represents a novel and effective therapeutic strategy for prevention and treatment of diabetic complications.

View larger version (46K): [in this window] [in a new window]   Figure 3. Schematic diagram of AGE-activated the Smad signaling pathway. Engagement of RAGE by AGEs activates Smad signaling directly via the ERK/p38 MAP kinase–Smad signaling cross-talk pathway and indirectly through the classic TGF-ß signaling pathway, also regulated by the ERK/p38 MAP kinases. Whereas ERK/p38 MAP kinase inhibitors and a neutralizing TGF-ß antibody block Smad signaling individually, overexpression of the inhibitory Smad7 blocks AGE-induced Smad signaling and diabetic complications generally.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1117fje

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