Hydroxysafflor Yellow A Ameliorates Renal Fibrosis by Suppressing TGF-β1-Induced Epithelial-to-Mesenchymal Transition

ObjectiveRenal fibrosis is the common pathological foundation of many chronic kidney diseases (CKDs). The aim of this study was to investigate whether Hydroxysafflor yellow A (HSYA) can preserve renal function by inhibiting the progression of renal fibrosis and the potential mechanisms.MethodsRenal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on 7-week-old C57BL/6 mice. HSYA (10, 50 and 100 mg/kg) were intragastrically administered. Sham group and model group were administered with the same volume of vehicle. Serum and kidney samples were collected 14 days after the UUO surgery. Serum biochemical indicators were measured by automatic biochemical analyzer. Histological changes were evaluated by HE and Masson staining. In vitro, the anti-fibrotic effect of HSYA was tested on human recombinant transforming growth factor-β1 (TGF-β1) stimulated HK-2 cells. The protein levels of α-SMA, collagen-I and fibronectin in kidney tissue andHK-2 cells were measured by immunohistochemistry and immunofluorescence. The protein levels of apoptosis-relative and TGF-β1/Smad3 signaling were detected by western blot.ResultsHSYA slowed the development of renal fibrosis both in vivo and in vitro. In UUO rats, renal function index suggested that HSYA treatment decreased the level of serum creatinine (Scr) and blood urea nitrogen (BUN) rose by UUO (P<0.05). HE staining and Masson staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration were notably attenuated in the high-dose HSYA group compared with the model group. The expressions of α-SMA, collagen-I and fibronectin were decreased in the UUO kidney and HK-2 cells of the HSYA-treatment group. Moreover, HSYA reduced the apoptotic rate of HK-2 cells stimulated by TGF-β1. Further study revealed that HSYA regulated the TGF-β1/Smads signaling pathway both in kidney tissue and HK-2 cells.ConclusionsThese results suggested that HSYA had a protective effect against fibrosis in renal cells, at least partly, through inhibiting TGF-β1/smad3-mediated Epithelial–mesenchymal transition signaling pathway.     

ShcA Protects against Epithelial–Mesenchymal Transition through Compartmentalized Inhibition of TGF-β-Induced Smad Activation

Epithelial–mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-β (TGF-β) signaling, and TGF-β drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-β also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-β type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-β-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-β receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-β/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-β receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-β receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-β signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-β-induced Smad activation through differential partitioning of receptor complexes at the cell surface.     

Loss of α-Tubulin Acetylation Is Associated with TGF-β-induced Epithelial-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.     

Hirsutella sinensis Attenuates Aristolochic Acid-Induced Renal Tubular Epithelial-Mesenchymal Transition by Inhibiting TGF-β1 and Snail Expression

Objective

To investigate the inhibitory effect of Hirsutella sinensis (HS) on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells induced by aristolochic acid (AA) and its possible mechanism.

Methods

18 male Sprague-Dawley rats were randomly and equally divided into the following 3 groups: AA group, AA+HS group and control group. Urinary protein excretion and creatinine clearance (CCr) were measured. All rats were sacrificed at the end of 12th week. The pathological examination of renal tissue was performed and the mRNA and protein expression of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), cytokeratin-18 and Snail in renal cortex were determined by real time quantitative PCR and immunohistochemical staining respectively. In addition, human renal proximal tubule epithelial cells line (HKC) was divided into the following 4 groups: AA group, AA+HS group, HS control group and control group. The above mRNA and protein expression in HKC was determined by real time quantitative PCR and Western blot respectively.

Results

(1) CCr was significantly decreased, and the urinary protein excretion and relative area of renal interstitial fibrosis were significantly increased in the rats of AA and AA+HS group compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly lightened in the rats of AA+HS group compared to those in AA group (P<0.05). (2) The mRNA and protein expression of TGF-β1, α-SMA and Snail was significantly up-regulated and the expression of cytokeratin-18 was significantly down-regulated in the rat renal cortex as well as in the cultured HKC cells in AA and AA+HS groups compared to those in control group (P<0.05 or P<0.01); all the above abnormalities significantly alleviated in AA+HS group compared to those in AA group (P<0.05 or P<0.01). (3) Knockdown endogenous Snail expression by siRNA could ameliorate AA-induced EMT of HKC cells, while overexpression of Snail by plasmid transfection diminished the antagonistic effect of HS on AA-induced EMT. These results suggest Snail might be a potential target of HS effect.

Conclusion

HS is able to antagonize, to some extent, tubular EMT and renal interstitial fibrosis caused by AA, which might be related to its inhibitory effects on the TGF-β1 and Snail expression.     

Combination of Active Components of Xiexin Decoction Ameliorates Renal Fibrosis Through the Inhibition of NF-κB and TGF-β1/Smad Pathways in db/db Diabetic Mice

Xiexin decoction, a herbal therapeutic agent commonly used in traditional Chinese medicine, is recognized for its beneficial effects on diabetic nephropathy exerted through the combined action of multiple components, including Rhizoma Coptidis alkaloids (A), Radix et Rhizoma Rhei polysaccharides (P), and Radix Scutellaria flavones (F). Our previous studies have shown that a combination of A, P, and F (APF) exhibits renoprotective effects against diabetic nephropathy. This study was aimed at determining the effects of APF on renal fibrosis in diabetic nephropathy and elucidating the underlying molecular mechanisms. To evaluate the effects of APF, in vivo, db/db diabetic mice were orally administered a low or high dose of APF (300 or 600 mg/kg, respectively) once a day for 8 weeks. We evaluated the blood and urine indices of metabolic and renal function, renal tissue histopathology, renal inflammation, and fibrosis. APF treatment significantly ameliorated glucose and lipid metabolism dysfunction, decreased urinary albumin excretion, normalized creatinine clearance, and reduced the morphological changes in renal tissue. Additionally, APF administration in db/db diabetic mice reduced the elevated levels of renal inflammation mediators such as intercellular adhesion molecule-1, monocyte chemotactic protein-1, tumor necrosis factor-α, interleukin-1β, and active nuclear factor κB (NF-κB). APF treatment also reduced type I and IV collagen, transforming growth factor-β1 (TGF-β1), and TGF-β1 type II receptor expression levels, and decreased the phosphorylation of Smad2/3 in the kidneys of db/db diabetic mice. These results suggest that APF reduces renal fibrosis in diabetic nephropathy through the NF-κB and TGF-β1/Smad signaling pathways. In vitro, APF treatment reduced cell proliferation and protein expression of α-smooth muscle actin, collagen I, TGF-β1 and NF-κB in mesangial cells cultured with high glucose concentrations. Our findings indicate that treatment with multi-component herbal therapeutic formulations may be a useful approach for the treatment of diabetic nephropathy.     

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β1²⁵–[41-65] and TGF-β1³⁰–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl₄-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl₄-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.     

TNF-α Induces Epithelial-Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC. Mol Cancer Res; 10(8); 1109-19. ?2012 AACR.     

Inhibition of the K+ Channel KCa3.1 Reduces TGF-β1-Induced Premature Senescence,Myofibroblast Phenotype Transition and Proliferation of Mesangial Cells

Objective

K_Ca3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K_Ca3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

Methods & Materials

Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K_Ca3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K_Ca3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05.

Results

K_ca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G₀-G₁ phase and the expression of K_ca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01).

Conclusion

Targeted disruption of K_Ca3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.     

The Polyphenols (?)-Epigallocatechin-3-Gallate and Luteolin Synergistically Inhibit TGF-β-Induced Myofibroblast Phenotypes through RhoA and ERK Inhibition

The presence of reactive stroma, predominantly composed of myofibroblasts, is directly associated with and drives prostate cancer progression. We have previously shown that (−)-Epigallocatechin-3-gallate (EGCG), in the form of Polyphenon E, significantly decreases serum levels of HGF and VEGF in prostate cancer patients. Given that HGF and VEGF are secreted from surrounding tumor myofibroblasts, these observations suggested that EGCG may inhibit prostate cancer-associated myofibroblast differentiation. Herein, we demonstrate that micromolar combinations of EGCG and a second polyphenol, luteolin, synergistically inhibit TGF-β-induced myofibroblast phenotypes in prostate fibroblast cell lines, as observed primarily by potentiation of fibronectin expression. Functionally, EGCG and luteolin inhibited TGF-β-induced extracellular matrix contraction, an enhancer of tumor cell invasion. EGCG and luteolin inhibited downstream TGF-β-induced signaling, including activation of ERK and AKT, respectively, but mechanistically, only ERK appeared to be necessary for TGF-β-induced fibronectin expression. Furthermore, neither EGCG nor luteolin affected Smad signaling or nuclear translocation. Rho signaling was found to be necessary for TGF-β-induced fibronectin expression and EGCG and luteolin each reduced RhoA activation. Finally, EGCG and luteolin were shown to reverse TGF-β-induced fibronectin expression, implicating that these natural compounds may be useful not only in preventing but also in treating already activated myofibroblasts and the diseases they cause, including cancer. The ability of EGCG and luteolin to synergistically target myofibroblasts suggests that combined clinical use of these compounds could prevent or reverse cancer progression through targeting the tumor microenvironment, in addition to the tumor itself.     

Functional Implications of MicroRNA-215 in TGF-β1-Induced Phenotypic Transition of Mesangial Cells by Targeting CTNNBIP1

Mesangial cell (MC) phenotypic transition is crucial for the progression of diabetic nephropathy. A major stimulus mediating high glucose-induced MC phenotypic transition is TGF-β1. Our current study focuses on microRNA-215 (miR-215) and investigates its role in TGF-β1-mediated MC phenotypic transition. Using real-time quantitative PCR (qRT-PCR) and northern blotting, we determined that the miR-192/215 family is dramatically upregulated under diabetic conditions both in vitro and in vivo. Gain- and loss-of-function approaches demonstrated that miR-215 inhibition significantly inhibited TGF-β1-induced mouse mesangial cell (MMC) phenotypic transition, whereas miR-215 upregulation promoted MMC phenotypic transition. Interestingly, these changes were not detected in cells that were treated with TGF-β1 and miR-192 mimics or inhibitors. These results suggest that miR-215 participates in TGF-β1-induced MMC phenotypic transition. Luciferase reporter assays were used to identify whether catenin-beta interacting protein 1 (CTNNBIP1) is a direct target of miR-215, which was predicted by bioinformatic analysis. Mechanistic studies revealed that CTNNBIP1 suppresses Wnt/β-catenin signaling and that miR-215 promotes β-catenin activation and upregulates α-SMA and fibronectin expression in TGF-β1-treated MMCs by targeting CTNNBIP1. In addition, in vivo miR-215 silencing with a specific antagomir significantly increased CTNNBIP1 protein expression, resulting in reduced β-catenin activity and decreased α-SMA and fibronectin expression in db/db mouse kidney glomeruli. Taken together, our findings indicate that miR-215 plays an essential role in MC phenotypic transition by regulating the CTNNBIP1/β-catenin pathway, which is related to the pathogenesis of diabetic nephropathy.     

Role of Complement 3 in TNF-α-Induced Mesenchymal Transition of Renal Tubular Epithelial Cells In Vitro

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.     

Dimethylfumarate Attenuates Renal Fibrosis via NF-E2-Related Factor 2-Mediated Inhibition of Transforming Growth Factor-β/Smad Signaling

TGF-β plays a key role in the development of renal fibrosis. Suppressing the TGF-β signaling pathway is a possible therapeutic approach for preventing this disease, and reports have suggested that Nrf2 protects against renal fibrosis by inhibiting TGF-β signaling. This study examines whether dimethylfumarate (DMF), which stimulates Nrf2, prevents renal fibrosis via the Nrf2-mediated suppression of TGF-β signaling. Results showed that DMF increased nuclear levels of Nrf2, and both DMF and adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, alpha-smooth muscle actin (α-SMA), fibronectin and type 1 collagen expression in TGF-β-treated rat mesangial cells (RMCs) and renal fibroblast cells (NRK-49F). Additionally, DMF and Ad-Nrf2 repressed TGF-β-stimulated Smad3 activity by inhibiting Smad3 phosphorylation, which was restored by siRNA-mediated knockdown of Nrf2 expression. However, downregulation of the antioxidant response element (ARE)-driven Nrf2 target genes such as NQO1, HO-1 and glutathione S-transferase (GST) did not reverse the inhibitory effect of DMF on TGF-β-induced upregulation of profibrotic genes or extracellular matrix proteins, suggesting an ARE-independent anti-fibrotic activity of DMF. Finally, DMF suppressed unilateral ureteral obstruction (UUO)-induced renal fibrosis and α-SMA, fibronectin and type 1 collagen expression in the obstructed kidneys from UUO mice, along with increased and decreased expression of Nrf2 and phospho-Smad3, respectively. In summary, DMF attenuated renal fibrosis via the Nrf2-mediated inhibition of TGF-β/Smad3 signaling in an ARE-independent manner, suggesting that DMF could be used to treat renal fibrosis.     

Enhanced Expression of Integrin αvβ3 Induced by TGF-β Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-β-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor β (TGF-β) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-β1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvβ3 induced by TGF-β on TGF-β-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-β1 in MCF10A and MCF12A mammary epithelial cells. TGF-β1 markedly amplified integrin αvβ3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-β1-induced EMT requires enhanced levels of both integrin αvβ3 expression and FGFR1. Knockdown of β3 suppressed the enhancement by FGF1 of TGF-β1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-β1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvβ3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-β1-induced EMT in mammary epithelial cells.     

The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.     

Apelin Ameliorates TNF-α-Induced Reduction of Glycogen Synthesis in the Hepatocytes through G Protein-Coupled Receptor APJ

Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.     

HIF-1α Promotes Epithelial-Mesenchymal Transition and Metastasis through Direct Regulation of ZEB1 in Colorectal Cancer

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1α) is involved in cancer metastasis, chemotherapy and poor prognosis. We previously found that deferoxamine, a hypoxia-mimetic agent, induces epithelial-mesenchymal transition (EMT) in colorectal cancer. Therefore, here we explored a new molecular mechanism for HIF-1α contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1α with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1α directly binding to the proximal promoter of ZEB1 via hypoxia response element (HRE) sites thus increasing the transactivity and expression of ZEB1. In addition, inhibition of ZEB1 was able to abrogate the HIF-1α-induced EMT and cell invasion. HIF-1α expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1α and ZEB1 were positively associated with Vimentin, an important mesenchymal marker of EMT, whereas negatively associated with E-cadherin expression. These findings suggest that HIF-1α enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1α and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1α, suggesting a novel molecular mechanism for HIF-1α-inducing EMT and cancer metastasis.