The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.     

Dietary fiber enhances TGF-β signaling and growth inhibition in the gut

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.     

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling. Depletion of RNF12 dramatically reduced TGF-β/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-β family signaling.     

Cutting edge: in the absence of TGF-β signaling in T cells, fewer CD103+ regulatory T cells develop, but exuberant IFN-γ production renders mice more susceptible to helminth infection

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.     

AIMP1/p43 downregulates TGF-beta signaling via stabilization of smurf2

AIMP1 (also known as p43) is a factor associated with a macromolecular aminoacyl-tRNA synthetase (ARS) complex but also plays diverse regulatory roles in various physiological processes. Here, we report that AIMP1 negatively regulates TGF-β signaling via stabilization of Smurf2. TGF-β-dependent phosphorylation and nuclear localization of R-Smads, induction of target genes, and growth arrest were increased in AIMP1-deficient or -suppressed cells. In AIMP1-deficient or suppressed cells, the Smurf2 level was decreased. Various binding assays demonstrated the direction interaction of the C-terminal region of AIMP1 directly with the Smad7-binding region of Smurf2. The association of Smurf2 with Smad7 and its ubiquitination were inhibited by AIMP1, thereby protecting its autocatalytic degradation stimulated by Smad7. Thus, this work suggests the novel activity of AIMP1 as a component of negative feedback loop of TGF-β signaling.     

A 4.3 kb Smad7 promoter is able to specify gene expression during mouse development

Members of transforming growth factor-β (TGF-β) superfamily play important roles in diverse biological functions including early development. These extracellular factors exert their effects by interacting with membrane receptors followed by signal transduction by a group of Smad proteins. Smad7 is an inhibitory Smad protein that specifically antagonizes TGF-β and activin signaling. To characterize the developmental role of Smad7, a transgenic mouse model was generated using a 4.3 kb mouse Smad7 promoter driving β-galactosidase expression. In these mice, the Smad7 promoter defined a restrictive expression pattern of β-galactosidase in a tightly regulated temporal and spatial manner. The β-galactosidase gene was transiently expressed in the cardiovascular structures including heart cushion tissues and the endothelium of major arteries at E11.5 to E12.5. Through E12.5 to E17.5, β-galactosidase expression was prominently detected in the epithelium of developing cochlea and nasolacrimal duct. In addition, it was temporally expressed in trigeminal ganglion, the skeletal muscles surrounding major joints, primordium of the jaws, as well as genital tubercle. These studies indicated that the 4.3 kb Smad7 promoter contains sufficient regulatory elements to define controlled gene expression during mouse development.     

Zyxin Is a Transforming Growth Factor-β (TGF-β)/Smad3 Target Gene That Regulates Lung Cancer Cell Motility via Integrin α5β1

Although TGF-β acts as a tumor suppressor in normal tissues and in early carcinogenesis, these tumor suppressor effects are lost in advanced malignancies. Single cell migration and epithelial-mesenchymal transition (EMT), both of which are regulated by TGF-β, are critical steps in mediating cancer progression. Here, we sought to identify novel direct targets of TGF-β signaling in lung cancer cells and have indentified the zyxin gene as a target of Smad3-mediated TGF-β1 signaling. Zyxin concentrates at focal adhesions and along the actin cytoskeleton; as such, we hypothesized that cytoskeletal organization, motility, and EMT in response to TGF-β1 might be regulated by zyxin expression. We show that TGF-β1 treatment of lung cancer cells caused rapid phospho-Smad3-dependent expression of zyxin. Zyxin expression was critical for the formation and integrity of cell adherens junctions. Silencing of zyxin decreased expression of the focal adhesion protein vasodilator-activated phospho-protein (VASP), although the formation and morphology of focal adhesions remained unchanged. Zyxin-depleted cells displayed significantly increased integrin α5β1 levels, accompanied by enhanced adhesion to fibronectin and acquisition of a mesenchymal phenotype in response to TGF-β1. Zyxin silencing led to elevated integrin α5β1-dependent single cell motility. Importantly, these features are mirrored in the K-ras-driven mouse model of lung cancer. Here, lung tumors revealed decreased levels of both zyxin and phospho-Smad3 when compared with normal tissues. Our data thus demonstrate that zyxin is a novel functional target and effector of TGF-β signaling in lung cancer. By regulating cell-cell junctions, integrin α5β1 expression, and cell-extracellular matrix adhesion, zyxin may regulate cancer cell motility and EMT during lung cancer development and progression.     

TNF-α suppressed TGF-β-induced CTGF expression by switching the binding preference of p300 from Smad4 to p65

TGF-β regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-β has been reported to mediate stimulatory action of TGF-β-induced ECM. Although TNF-α was reported to suppress the TGF-β-induced CTGF gene expression, the molecular mechanism is not well clarified. In this study, we found the inhibitory effect of TNF-α on TGF-β-induced CTGF expression in WT but not p65?/? MEF cells. TNF-α neither induced Smad7 expression nor affected TGF-β-induced Smad2 phosphorylation and nuclear translocation. We demonstrated that p300 physically associated with p65 rather than Smad4 in the presence of both TNF-α and TGF-β. Moreover, the TGF-β-induced binding of p300 and acetylated H4, but not Smad4 to the CTGF promoter was disturbed by TNF-α treatment. Overall, our data showed that suppression of TNF-α on TGF-β-induced CTGF expression is due to the competition of p300 by p65 and Smad4.     

Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-β/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3-deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3(-)(/-) white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3(-/-) adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-1α expression. We observe significant correlation between TGF-β1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-β signaling protects mice from obesity, diabetes, and hepatic steatosis. Together, these results demonstrate that TGF-β signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-β activity might be an effective treatment strategy for obesity and diabetes.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

Protection of cerulein-induced pancreatic fibrosis by pancreas-specific expression of Smad7

Pancreatic fibrosis is the hallmark of chronic pancreatitis, currently an incurable disease. Pancreatitis fibrosis is caused by deposition of extracellular matrix (ECM) and the underlying pathological mechanism remains unclear. In addition to its broad biological activities, TGF-β is a potent pro-fibrotic factor and many in vitro studies using cell systems have implicated a functional role of TGF-β in the pathogenesis of pancreatic fibrosis. We analyzed the in vivo role of TGF-β pathway in pancreatic fibrosis in this study. Smad7, an intracellular inhibitory protein that antagonizes TGF-β signaling, was specifically expressed in the pancreas using a transgenic mouse model. Chronic pancreatitis was induced in the mouse with repeated administration of cerulein. Smad7 expression in the pancreas was able to significantly inhibit cerulein-induced pancreatic fibrosis. Consistently, the protein levels of collagen I and fibronectin were decreased in the Smad7 transgenic mice. In addition, α-smooth muscle actin, a marker of activated pancreas stellate cells, was reduced in the transgenic mice. Taken together, these data indicate that inhibition of TGF-β signaling by Smad7 is able to protect cerulein-induced pancreatic fibrosis in vivo.     

Paracrine TGF-β signaling counterbalances BMP-mediated repression in hair follicle stem cell activation

Hair follicle (HF) regeneration begins when communication between quiescent epithelial stem cells (SCs) and underlying mesenchymal dermal papillae (DP) generates sufficient activating cues to overcome repressive BMP signals from surrounding niche cells. Here, we uncover a hitherto unrecognized DP transmitter, TGF-β2, which activates Smad2/3 transiently in HFSCs concomitant with entry into tissue regeneration. This signaling is critical: HFSCs that cannot sense TGF-β exhibit significant delays in HF regeneration, whereas exogenous TGF-β2 stimulates HFSCs in vivo and in vitro. By engineering TGF-β- and BMP-reporter mice, we show that TGF-β2 signaling antagonizes BMP signaling in HFSCs but not through competition for limiting Smad4-coactivator. Rather, our microarray, molecular, and genetic studies unveil Tmeff1 as a direct TGF-β2/Smad2/3 target gene, expressed by activated HFSCs and physiologically relevant in restricting and lowering BMP thresholds in the niche. Connecting BMP activity to an SC's response to TGF-βs may explain why these signaling factors wield such diverse cellular effects.     

Smad4介导转化生长因子-β信号调节骨骼发育和稳态维持的功能

转化生长因子-β（TGF-β）是一个包括数十种TGF-βs、骨形态发生蛋白（BMPs）等配体在内的生长因子超家族,在哺乳动物整体和组织器官发育过程中具有广泛而重要的功能。Smad4是细胞内TGF-β信号通路的核心信号转导分子。为了深入研究Smad4介导的TGF-β信号在骨骼发育过程中的生理功能,我们利用转基因技术研制了软骨细胞、肥大型软骨细胞和成骨细胞分别特异性表达Cre重组酶的转基因小鼠,利用条件基因敲除技术研制了不同类型骨骼细胞Smad4基因敲除的小鼠模型。表型分析结果揭示了Smad4在软骨细胞增殖和分化、骨重塑以及稳态维持过程中的功能以及相关的分子机制,为理解人类相关骨骼疾病的发生及其机理提供了新的线索。     

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.