Jianlin Zhang

E-Cadherin/β-Catenin Complex and the Epithelial Barrier

E-Cadherin/β-catenin complex plays an important role in maintaining epithelial integrity and disrupting this complex affect not only the adhesive repertoire of a cell, but also the Wnt-signaling pathway. Aberrant expression of the complex is associated with a wide variety of human malignancies and disorders of fibrosis resulting from epithelial-mesenchymal transition. These associations provide insights into the complexity that is likely responsible for the fibrosis/tumor suppressive action of E-cadherin/β-catenin.

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage.

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial–mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0–3, 6–9 or 10–13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.

Association of β-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-β1.

Summary Transforming growth factor &bgr;1 (TGF-&bgr;1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-&bgr;/Smad and Wnt/&bgr;-catenin pathways in epithelial–mesenchymal transition (EMT) suggests a specific role for &bgr;-catenin in profibrotic effects of TGF-&bgr;1. However, no such mechanistic role has been demonstrated for &bgr;-catenin in the anti-inflammatory effects of TGF-&bgr;1. In the present study, we explored the role of &bgr;-catenin in the profibrotic and anti-inflammatory effects of TGF-&bgr;1 by using a cytosolic, but not membrane, &bgr;-catenin knockdown chimera (F-TrCP-Ecad) and the &bgr;-catenin/CBP inhibitor ICG-001. TGF-&bgr;1 induced nuclear Smad3/&bgr;-catenin complex, but not &bgr;-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-&bgr;1-induced cytoplasmic &bgr;-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-&bgr;1-induced Smad3/&bgr;-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-&bgr;1-induced EMT depends on &bgr;-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the &bgr;-catenin level was low and was not changed by interferon-&ggr; (IFN-&ggr;) or lipopolysaccharide (LPS) with or without TGF-&bgr;1. TGF-&bgr;1 inhibition of LPS-induced TNF-&agr; and IFN-&ggr;-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type &bgr;-catenin in J774 cells. Inhibition of &bgr;-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-&bgr;1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that &bgr;-catenin is required as a co-factor of Smad in TGF-&bgr;1-induced EMT of C1.1 epithelial cells, but not in TGF-&bgr;1 inhibition of macrophage activation. Targeting &bgr;-catenin may dissociate the TGF-&bgr;1 profibrotic and anti-inflammatory effects.

Matrix metalloproteinase 9-dependent Notch signaling contributes to kidney fibrosis through peritubular endothelial-mesenchymal transition.

ABSTRACT Background: Endothelial cells are known to contribute to kidney fibrosis via endothelial–mesenchymal transition (EndoMT). Matrix metalloproteinase 9 (MMP-9) is known to be profibrotic. However, whether MMP-9 contributes to kidney fibrosis via EndoMT is unknown. Methods: Primary mouse renal peritubular endothelial cells (MRPECs) were isolated and treated by recombinant human transforming growth factor beta 1 (rhTGF-β1) with or without MMP-9 inhibitor or by recombinant human MMP-9 (rhMMP-9) alone. Kidney fibrosis was induced by unilateral ureteral obstruction (UUO) in MMP-9 knockout (KO) and wide-type (WT) control mice. The effects of MMP-9 on EndoMT of MRPECs and kidney fibrosis were examined. Results: We showed that MRPECs underwent EndoMT after rhTGF-β1 treatment or in UUO kidney as evidenced by decreased expression of endothelial markers, vascular endothelial cadherin (VE-cadherin) and CD31, and increased levels of mesenchymal markers, α-smooth muscle actin (α-SMA) and vimentin. The expression of fibrosis markers was also up-regulated significantly after rhTGF-β1 treatment in MRPECs. The EndoMT and fibrosis markers were significantly less in rhTGF-β1-treated MMP-9 KO MRPECs, whereas MMP-9 alone was sufficient to induce EndoMT in MRPECs. UUO kidney of MMP-9 KO mice showed significantly less interstitial fibrosis and EndoMT in MRPECs. Notch signaling shown by Notch intracellular domain (NICD) was increased, while Notch-1 was decreased in rhTGF-β1-treated MRPECs of MMP-9 WT but not MMP-9 KO mice. Inhibition of MMP-9 or Notch signaling prevented rhTGF-β1- or rhMMP-9-induced α-SMA and NICD upregulation in MRPECs. UUO kidney of MMP-9 KO mice had less staining of Notch signaling transcription factor Hey-1 in VE-cadherin-positive MRPECs than WT controls. Conclusions: Our results demonstrate that MMP-9-dependent Notch signaling plays an important role in kidney fibrosis through EndoMT of MRPECs.

Redirecting TGF-βSignaling through theβ-Catenin/Foxo Complex Prevents Kidney Fibrosis

TGF-β is a key profibrotic factor, but targeting TGF-β to prevent fibrosis also abolishes its protective anti-inflammatory effects. Here, we investigated the hypothesis that we can redirect TGF-β signaling by preventing downstream profibrotic interaction of β-catenin with T cell factor (TCF), thereby enhancing the interaction of β-catenin with Foxo, a transcription factor that controls differentiation of TGF-β induced regulatory T cells (iTregs), and thus, enhance anti-inflammatory effects of TGF-β In iTregs derived from EL4 T cells treated with recombinant human TGF-β1 (rhTGF-β1) in vitro, inhibition of β-catenin/TCF transcription with ICG-001 increased Foxp3 expression, interaction of β-catenin and Foxo1, binding of Foxo1 to the Foxp3 promoter, and Foxo transcriptional activity. Moreover, the level of β-catenin expression positively correlated with the level of Foxo1 binding to the Foxp3 promoter and Foxo transcriptional activity. T cell fate mapping in Foxp3gfp Ly5.1/5.2 mice revealed that coadministration of rhTGF-β1 and ICG-001 further enhanced the expansion of iTregs and natural Tregs observed with rhTGF-β1 treatment alone. Coadministration of rhTGF-β1 with ICG-001 also increased the number of Tregs and reduced inflammation and fibrosis in the kidney fibrosis models of unilateral ureteric obstruction and ischemia-reperfusion injury. Notably, ICG-001 prevented the fibrosis in distant organs (lung and liver) caused by rhTGF-β1. Together, our results show that diversion of β-catenin from TCF- to Foxo-mediated transcription inhibits the β-catenin/TCF-mediated profibrotic effects of TGF-β while enhancing the β-catenin/Foxo-mediated anti-inflammatory effects. Targeting β-catenin/Foxo may be a novel therapeutic strategy in the treatment of fibrotic diseases that lead to organ failure.