Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation in vivo and inhibited fibroblast activation in vitro. HDAC5 knockdown (KD) significantly downregulated TGF-β1 induced Smad2/3 phosphorylation and increased Smad7 expression. Meanwhile, Smad7 KD rescued the Smad2/3 phosphorylation downregulation and scar hyperplasia inhibition mediated by HDAC5 deficiency. Luciferase reporter assays and ChIP-qPCR assays revealed that HDAC5 interacts with myocyte enhancer factor 2A (MEF2A) suppressing MEF2A binding to the Smad7 promoter region, which results in Smad7 promoter activity repression. HDAC4/5 inhibitor, LMK235, significantly alleviated hypertrophic scar formation. Our study provides clues for the development of HDAC5 targeting strategies for the therapy or prophylaxis of fibrotic diseases.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

Brightfield Proximity Ligation Assay Reveals Both Canonical and Mixed Transforming Growth Factor-β/Bone Morphogenetic Protein Smad Signaling Complexes in Tissue Sections

Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3–Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15–16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.     

Itch E3 Ubiquitin Ligase Positively Regulates TGF-β Signaling to EMT via Smad7 Ubiquitination

TGF-β regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-β-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-β signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-β receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-β signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-β signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-β signaling and of TGF-β-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.     

Smad3 Regulates Rho Signaling via NET1 in the Transforming Growth Factor-β-induced Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells

We previously demonstrated that RhoA-dependent signaling regulates transforming growth factor-β1 (TGF-β1)-induced cytoskeletal reorganization in the human retinal pigment epithelial cell line ARPE-19. Smad pathways have also been shown to mediate TGF-β1 activity. Here, we examined what regulates Rho GTPase activity and tested whether Smad signaling cross-talks with Rho pathways during TGF-β1-induced actin rearrangement. Using small interfering RNAs, we found that NET1, the guanine nucleotide exchange factor of RhoA, is critical for TGF-β1-induced cytoskeletal reorganization, N-cadherin expression, and RhoA activation. In ARPE-19 cells lacking NET1, TGF-β1-induced stress fibers and N-cadherin expression were not observed. Interestingly, in dominant-negative Smad3-expressing or constitutively active Smad7 cells, TGF-β1 failed to induce NET1 mRNA and protein expression. Consistent with these results, both dominant-negative Smad3 and constitutively active Smad7 blocked the cytoplasmic localization of NET1 and inhibited interactions between NET1 and RhoA. Finally, we found that NET1 is a direct gene target of TGF-β1 via Smad3. Taken together, our results demonstrate that Smad3 regulates RhoA activation and cytoskeletal reorganization by controlling NET1 in TGF-β1-induced ARPE-19 cells. These data define a new role for Smad3 as a modulator of RhoA activation in the regulation of TGF-β1-induced epithelial-mesenchymal transitions.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.