Response gene to complement 32 is essential for fibroblast activation in renal fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

Divergence and convergence of TGF-beta/BMP signaling

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.     

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.     

Interleukin-7 and transforming growth factor-beta play counter-regulatory roles in protein kinase C-delta-dependent control of fibroblast collagen synthesis in pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic factor responsible for promoting synthesis of extracellular matrix. Interleukin-7 (IL-7) inhibits TGF-beta signaling by up-regulating Smad7, a major inhibitor of the Smad family. In a variety of cells, TGF-beta-mediated activation of target genes requires active protein kinase C-delta (PKC-delta) in addition to Smads (1). We determined the role of PKC-delta in the regulation of pulmonary fibroblast collagen synthesis in response to TGF-beta and IL-7 stimulation. Here we show that TGF-beta and IL-7 have opposing effects on PKC-delta; TGF-beta stimulates, while IL-7 inhibits, PKC-delta activity. IL-7 inhibits TGF-beta-induced PKC-delta phosphorylation at Ser-645 and Thr-505. Inhibition of PKC-delta with specific small inhibitory RNA restores TGF-beta-mediated induction of Smad7 and in parallel significantly reduces TGF-beta-mediated collagen synthesis. Thus, PKC-delta may play a critical role in the pathogenesis of pulmonary fibrosis and may serve as a molecular target for therapeutic intervention to suppress fibrosis.     

Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation

TGF-beta inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-beta in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-beta signaling in adipocyte differentiation, we characterized the expression of the TGF-beta receptors, and the Smads which transmit or inhibit TGF-beta signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-beta receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-beta responses, decreased severely. Dominant negative interference with TGF-beta receptor signaling, by stably expressing a truncated type II TGF-beta receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-beta-induced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-beta was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-beta signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-beta responses, enhance the effects of TGF-beta on these cells.     

TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses

Through the action of its membrane-bound type I receptors, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of the signaling responses induced by TGF-beta are mediated by Smad proteins, but certain evidence has suggested that TGF-beta can also signal independently of Smads. We found in mouse mammary epithelial (NMuMG) cells, which respond to TGF-beta treatment in multiple ways, that TGF-beta-induced activation of p38 MAP kinase is required for TGF-beta-induced apoptosis, epithelial-to-mesenchymal transition (EMT), but not growth arrest. We further demonstrated that activation of p38 is independent of Smads using a mutant type I receptor, which is incapable of activating Smads but still retains the kinase activity. This mutant receptor is sufficient to activate p38 and cause NMuMG cells to undergo apoptosis. However, it is not sufficient to induce EMT. These results indicate that TGF-beta receptor signals through multiple intracellular pathways and provide first-hand biochemical evidence for the existence of Smad-independent TGF-beta receptor signaling.