Adipocytes recognize and degrade oxidized low density lipoprotein through CD36

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in controlling critical cellular activities including proliferation, differentiation, extracellular matrix production, and apoptosis. TGF-beta signals are mediated by a family of Smad proteins, of which Smad2 and Smad3 are downstream intracellular targets of serine/threonine kinase receptors of TGF-beta. Although Smad2 and Smad3 are crucial for TGF-beta signaling, little is known about the regulation of their expression. In this study, we investigated the expression of Smad2 and Smad3 in an in vivo animal model of lung fibrosis induced by bleomycin. We found that the expression of Smad3 was regulated in lungs during bleomycin-induced pulmonary fibrosis. The decline of Smad3 mRNA was evident at day three of post-bleomycin instillation and the expression of Smad3 continually decreased during the reparative phase of lung injury (days 8 and 12), whereas the expression of Smad2 showed little change after bleomycin administration. We further investigated whether the expression of Smad3 was regulated by TGF-beta in an in vitro lung fibroblast culture system. Our results show an immediate translocation of Smad3 protein from the cytoplasm to the nucleus and a delayed down-regulation of Smad3 mRNA by TGF-beta in lung fibroblasts. These studies provide direct evidence for a differential regulation of Smad3 expression that is distinct from that of Smad2 during bleomycin-induced pulmonary fibrosis and suggest a ligand-induced negative feedback loop that modulates cellular TGF-beta signaling.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

三七皂苷R1 对肝纤维化大鼠TGF-beta1/Smad3信号的影响

目的:探讨SD大鼠肝纤维化后肝组织及血清中转化生长因子-β1(Transforming Growth Factor-β1,TGF-β1)及Smad3的表达和变化,以及三七皂苷R1对肝纤维化的保护作用。方法:72只健康雄性SD大鼠分为对照组、二甲基亚硝胺(NDMA)组和三七皂苷R1组,再按不同时间点分为1、2、4周,3个亚组,每个亚组8只动物。NDMA组采用NDMA 2 m L/kg腹腔注射,三七皂苷R1组同时静脉注射三七皂苷R1,剂量为100 mg/kg体重,对照组注射等量的生理盐水。在各组的不同时间点采用RT-PCR及ELISA技术检测肝组织及血清中TGF-β1、Smad3的表达及变化。结果:1、TGF-β1、Smad3 m RNA及蛋白在各组中均有表达。2、对照组各时间点比较均无统计学意义(P>0.05)。NDMA组中,随着损伤时间的延长,TGF-β1、Smad3 m RNA及蛋白的表达逐渐上调,且各时间点与对照组比较有统计学意义(P<0.05)。而三七皂苷R1组TGF-β1、Smad3 m RNA及蛋白在各时间点均较NDMA组表达下调,有统计学意义(P<0.05)。结论:1、TGF-β1/Smad3信号参与了肝纤维化的发生和发展过程,且随损伤的逐渐加重,表达越高。2、三七皂苷R1可降低肝组织中TGF-β1/Smad3信号的表达,减轻肝细胞的纤维化,发挥保护肝组织损伤的作用。     

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.     

PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.     

Decreased Smad 7 expression contributes to cardiac fibrosis in the infarcted rat heart

We examined the role of the transforming growth factor (TGF)-beta(1) signaling inhibitor Smad 7 in cardiac fibrosis. TGF-beta(1) (10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-beta type I receptor (TbetaRI), and TGF-beta(1) proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TbetaRI expression were decreased in scar tissue, whereas TGF-beta(1) expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.     

Smad4 is essential for down-regulation of E-cadherin induced by TGF-beta in pancreatic cancer cell line PANC-1

Smad4 is a tumour suppressor gene frequently deleted in pancreatic cancer. To investigate the roles of Smad4 deficiency in invasive and matastatic capabilities of pancreatic cancer, we examined the effects of Smad4 deficiency on regulation of the invasion suppressor E-cadherin in pancreatic cancer cell line PANC-1. TGF-beta decreased expression of E-cadherin and beta-catenin proteins at the plasma membrane, increased Snail and Slug mRNA expression, and induced fibroblastoid morphology in PANC-1 cells. These effects of TGF-beta were abrogated in Smad4-knocked-down PANC-1 cells. We also found that TGF-beta-induced down-regulation of E-cadherin expression was partially inhibited in Snail- and Slug-knocked-down PANC-1 cells. Thus, Smad4 mediates down-regulation of E-cadherin induced by TGF-beta in PANC-1 cells, at least in part, through Snail and Slug induction. These results suggest that Smad4 deficiency observed in invasive and metastatic pancreatic cancer might not be linked to the loss of E-cadherin.     

Epithelial to mesenchymal transition in Madin-Darby canine kidney cells is accompanied by down-regulation of Smad3 expression,leading to resistance to transforming growth factor-beta-induced growth arrest

In normal epithelial cells, transforming growth factor-beta (TGF-beta) typically causes growth arrest in the G(1) phase of the cell cycle and may eventually lead to apoptosis. However, transformed cells lose these inhibitory responses and often instead show an increase in malignant character following TGF-beta treatment. In the canine kidney-derived epithelial cell line, MDCK, synergism between activation of the Raf/MAPK pathway and the resulting autocrine production of TGF-beta triggers transition from an epithelial to a mesenchymal phenotype. During this process, these cells become refractive to TGF-beta-induced cell cycle arrest and apoptosis. TGF-beta signals are primarily transduced to the nucleus through complexes of receptor-regulated Smads, Smad2 and Smad3 with the common mediator Smad, Smad4. Here we show that the transition from an epithelial to mesenchymal phenotype is accompanied by gradual down-regulation of expression of Smad3. Restoration of Smad3 to previous levels of expression restores the cell cycle arrest induced by TGF-beta without reverting the cells to an epithelial phenotype or impacting on the MAPK pathway. Regulation of apoptosis is not affected by Smad3 levels. These data attribute to Smad3 a critical role in the control of cell proliferation by TGF-beta, which is lost following an epithelial to mesenchymal transition.     

Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins

This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Epigallocatechin-3-Gallate Attenuates Unilateral Ureteral Obstruction-Induced Renal Interstitial Fibrosis in Mice

The severity of tubulointerstitial fibrosis is regarded as an important determinant of renal prognosis. Therapeutic strategies targeting tubulointerstitial fibrosis have been considered to have potential in the treatment of chronic kidney disease. This study aims to evaluate the protective effects of (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, against renal interstitial fibrosis in mice. EGCG was administrated intraperitoneally for 14 days in a mouse model of unilateral ureteral obstruction (UUO). The results of our histological examination showed that EGCG alleviated glomerular and tubular injury and attenuated renal interstitial fibrosis in UUO mice. Furthermore, the inflammatory responses induced by UUO were inhibited, as represented by decreased macrophage infiltration and inflammatory cytokine production. Additionally, the expression of type I and III collagen in the kidney were reduced by EGCG, which indicated an inhibition of extracellular matrix accumulation. EGCG also caused an up-regulation in α-smooth muscle actin expression and a down-regulation in E-cadherin expression, indicating the inhibition of epithelial-to-mesenchymal transition. These changes were found to be in parallel with the decreased level of TGF-β1 and phosphorylated Smad. In conclusion, the present study demonstrates that EGCG could attenuate renal interstitial fibrosis in UUO mice, and this renoprotective effect might be associated with its effects of inflammatory responses alleviation and TGF-β/Smad signaling pathway inhibition.     

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.     

Functional role and species-specific contribution of arginases in pulmonary fibrosis

Lung fibrosis is characterized by increased deposition of ECM, especially collagens, and enhanced proliferation of fibroblasts. l-arginine is a key precursor of nitric oxide, asymmetric dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that l-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant, time-dependent decrease in pulmonary l-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, compared with controls, and assessed by qRT-PCR and Western blot analysis. TGF-beta1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH/3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, NG-hydroxy-l-arginine, attenuated TGF-beta1-stimulated collagen deposition, but not collagen mRNA expression or Smad signaling, in fibroblasts. In human lungs derived from patients with idiopathic pulmonary fibrosis, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-beta1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and l-arginine metabolism during the course of lung fibrosis in the mouse, whereas species-specific regulatory mechanisms may account for the differences observed in mouse and human.