TRANSFORMING GROWTH FACTOR-β1 AND SMAD4 SIGNALING PATHWAY DOWN-REGULATES RENAL EXTRACELLULAR MATRIX DEGRADATION IN DIABETIC RATS

Objective To investigate the role of transforming growth factor-131 （TGF-β1）/Smad4 pathway in development of renal fibrosis in streptozotocin （STZ）-induced diabetic nephropathy （DN） rats and explore its possible mechanism. Methods Male Wistar rats weighing 180-220 g were divided into 5 groups： group A （ normal control）, group B [ diabetes mellitus （DM） 2 weeks ], group C （ DM 4 weeks）, group D （ DM 8 weeks）, and group E （ DM 16 weeks）. Except for the normal control group, other groups were induced DM by single injection of STZ （55 mg/kg） respectively. Blood glucose level, serum creatinine, and 24-hour urine protein were examined. Expressions of TGF-β1 and Smad4 protein and mRNA in kidney were detected using immunohistochemical technique, Western blot, and real-time PCR. mRNA expressions of stromelysin-1 （ MMP-3 ）, tissue inhibitor of metalloproteinase-1 （ TIMP-1 ）, and collagen Ⅲ in kidney were also detected by real-time PCR. Results The levels of blood glucose, serum creatinine, and 24-hour urine protein in rats of group B, C, D, and E were higher than those of the control group. With the progression of renal fibrosis, the expressions of TGF-β1 and Smad4 protein and mRNA in kidney of diabetic rats elevated. In addition, the renal MMP-3 mRNA expression diminished in diabetic rats, while TIMP-1 and collagen Ⅲ mRNA increased. Conclusions In STZ-induced diabetic rats, the TGF-β1/Smad4 appears to play an important role in renal fibrosis of DN. The increased expression of TGF-β1 and Smad4 might result in the transcriptional regulation of downstream target genes of TGF-β1/Smad4 pathway, which contributes to the progression of renal fibrosis in diabetic rats.

Roles of transforming growth factor-βsignaling in liver disease

In this editorial we expand the discussion on the article by Zhang et al published in the recent issue of the World Journal of Hepatology.We focus on the diagnostic and therapeutic targets identified on the basis of the current understanding of the molecular mechanisms of liver disease.Transforming growth factor-β(TGF-β)belongs to a structurally related cytokine super family.The family members display different time-and tissue-specific expression patterns associated with autoimmunity,inflammation,fibrosis,and tumorigenesis;and,they participate in the pathogenesis of many diseases.TGF-βand its related signaling pathways have been shown to participate in the progression of liver diseases,such as injury,inflammation,fibrosis,cirrhosis,and cancer.The often studied TGF-β/Smad signaling pathway has been shown to promote or inhibit liver fibrosis under different circumstances.Similarly,the early immature TGF-βmolecule functions as a tumor suppressor,inducing apoptosis;but,its interaction with the mitogenic molecule epidermal growth factor alters this effect,activating anti-apoptotic signals that promote liver cancer development.Overall,TGF-βsignaling displays contradictory effects in different liver disease stages.Therefore,the use of TGF-βand related signaling pathway molecules for diagnosis and treatment of liver diseases remains a challenge and needs further study.In this editorial,we aim to review the evidence for the use of TGF-βsignaling pathway molecules as diagnostic or therapeutic targets for different liver disease stages.

Dab2 attenuates brain injury in APP/PS1 mice via targeting transforming growth factor-beta/SMAD signaling

Transforming growth factor-beta （TGF-β） type II receptor （TβRⅡ） levels are extremely low in the brain tissue of patients with Alzheimer＇s disease. This receptor inhibits TGF-β1/SMAD signaling and thereby aggravates amyolid-beta deposition and neuronal injury. Dab2, a specific adapter protein, protects T RII from degradation and ensures the effective conduction of TGF-β 1/SMAD signaling. In this study, we used an adenoviral vector to overexpress the Dab2 gene in the mouse hippocampus and investigated the regulatory effect of Dab2 protein on TGF-β1/SMAD signaling in a mouse model of Alzheimer＇s disease, and the potential neuroprotective effect. The results showed that the TβRⅡ level was lower.in APP/PS1 mouse hippocampus than in normal mouse hippocampus. After Dab2 expression, hippocampal TβRⅡ and p-SMAD2/3 levels were signifi- cantly increased, while amyloid-beta deposition, microglia activation, tumor necrosis factor- and interleulin-6 levels and neuronal loss were significantly attenuated in APP/PS1 mouse brain tissue. These results suggest that Dab2 can exhibit neuroprotective effects in Alzheimer＇s disease by regulating TGF-β1/SMAD signaling.

miR-34a mediates oxaliplatin resistance of colorectal cancer cells by inhibiting macroautophagy via transforming growth factor-β/Smad4 pathway

To investigate whether microRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colorectal cancer (CRC) cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway.METHODSmiR-34a expression levels were detected in CRC tissues and CRC cell lines by quantitative real-time polymerase chain reaction. Computational search, functional luciferase assay and western blotting were used to demonstrate the downstream target of miR-34a in CRC cells. Cell viability was measured with Cell Counting Kit-8. Apoptosis and macroautophagy of CRC cells were analyzed by flow cytometry and transmission electron microscopy, and expression of beclin I and LC3-II was detected by western blotting.RESULTSExpression of miR-34a was significantly reduced while expression of TGF-β and Smad4 was increased in CRC patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a levels and increased TGF-β and Smad4 levels in both parental cells and the OXA-resistant CRC cells. Activation of macroautophagy contributed to OXA resistance in CRC cells. Expression levels of Smad4 and miR-34a in CRC patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in CRC cells.CONCLUSIONmiR-34a mediates OXA resistance of CRC by inhibiting macroautophagy via the TGF-β/Smad4 pathway.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

Tetrandrine inhibits activation of rat hepatic stellate cells in vitro via transforming growth factor-β signaling

AIM: To investigate the effect of various concentrations of tetrandrine on activation of quiescent rat hepatic stellate cells (HSCs) and transforming growth factor-β (TGF-β) signaling in vitro.METHODS: HSCs were isolated from rats by in situperfusion of liver and 18% Nycodenz gradient centrifugation, and primarily cultured on uncoated plastic plates for 24 hwith DMEM containing 20% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, the HSCs were cultured in 2% FBS/DMEM with tetrandrine (0.25, 0.5, 1,2 mg/L, respectively). Cell morphological features were observed under an inverted microscope, smooth muscleα-actin (α-SMA) was detected by immunocytochemistry and image analysis system, laminin (LN) and type Ⅲprocollagen (PCⅢ) in supernatants were determined byradioimmunoassay. TGF-β1 mRNA, Smad 7 mRNA and Smad 7 protein were analyzed with RT-PCR and Western blotting, respectively.RESULTS: Tetrandrine at the concentrations of 0.25-2 mg/L prevented morphological transformation of HSC from the quiescent state to the activated one, while α-SMA, LN and PCⅢ expressions were inhibited. As estimated by gray values, the expression of α-SMA in tetrandrine groups (0.25, 0.5, 1, 2 mg/L) was reduced from 21.3% to 42.2%(control: 0.67, tetrandrine groups: 0.82, 0.85, 0.96, or 0.96, respectively, which were statistically different from the control, P＜0.01), and the difference was more significant in tetrandrine at 1 and 2 mg/L. The content of LN in supernatants was significantly decreased in tetrandrine groups to 58.5%, 69.1%, 65.8% or 60.0% that of the control respectively, and that of PCⅢ to 84.6%, 81.5%,75.7% or 80.7% respectively (P＜0.05 vs control), with no significant difference among tetrandrine groups. RTPCR showed that TGF-β1 mRNA expression was reduced by tetrandrine treatments from 56.56% to 87.90% in comparison with the control, while Smad 7 mRNA was increased 1.4-4.8 times. The TGF-β1 mRNA and Smad 7 mRNA expression was in a significant negative correlation (r= -0.755, P＜0.01), and both were significantly correlated with α-SMA protein expression (r = -0.938, P＜0.01;r = 0.938, P＜0.01, respectively). The up-regulation of Smad 7 protein by tetrandrine (1 mg/L)was confirmed by Western blotting as well.CONCLUSION: Tetrandrine has a direct inhibiting effect on the activation of rat HSCs in culture. It up-regulates the expression of Smad 7 which in turn blocks TGF-β1 expression and signaling.

Regulation of transforming growth factor-β signaling as a therapeutic approach to treating colorectal cancer

According to data from 2020,Slovakia has long been among the top five countries with the highest incidence rate of colorectal cancer(CRC)worldwide,and the rate is continuing to rise every year.In approximately 80%of CRC cases,allelic loss(loss of heterozygosity,LOH)occurs in the long arm of chromosome 18q.The most important genes that can be silenced by 18q LOH or mutations are small mothers against decapentaplegic homolog(SMAD)2 and SMAD4,which are intracellular mediators of transforming growth factor(TGF)-βsuperfamily signals.TGF-βplays an important role in the pro-oncogenic processes,including such properties as invasion,epithelial-mesenchymal transition(commonly known as EMT),promotion of angiogenesis,and immunomodulatory effects.Several recent studies have reported that activation of TGF-βsignaling is related to drug resistance in CRC.Because the mechanisms of drug resistance are different between patients in different stages of CRC,personalized treatment is more effective.Therefore,knowledge of the activation and inhibition of factors that affect the TGF-βsignaling pathway is very important.

Effects of the Nocth signaling pathway on expression of inflammatory factors and transforming growth factors in diabetic foot ulcers

Objective:persistent hyperinflammation is an important reason for the development of diabetic foot ulcer.Notch signaling is an important signaling pathway involved in the inflammatory response and cell proliferation in diabetic foot ulcer rats.This paper aims to explore the effect of Notch signaling on inflammatory factors,chemokines and growth factors through the intervention of Notch signaling in diabetic foot ulcer rats.Methods:the experimental model was made by using high-fat feed combined with streptozotocin(STZ)to cause diabetes,and the experimental model of diabetic foot ulcer was established by constant temperature and constant pressure scald apparatus.The normal ulcer model was used as a control.The intervention controls of the experimental model included normal saline,western medicine growth factor,Notch agonist Jagged1,Notch signaling inhibitor ly-411575,and the intervention of traditional Chinese medicine Zizhu ointment for 7 days.Serum il-1,il-6,TNF-radiation,and il-17 were detected by ELISA.Real-time PCR was used to detect the inflammatory factors,chemokines,and growth factors associated with Notch signaling in wound tissues:tnf-uum,il-1,il-6,il-17,interleukin-8,ip-10,McP-1,TGF-uum,TGF-livelihood.Results:serum levels of il-1,il-6,TNF-radiation and il-17 in diabetic foot ulcer rats were significantly higher than that in normal ulcer rats.The contents of il-1,il-6,TNF-radiation and il-17a in ly-411575 group and Zizhu ointment group were significantly reduced.Real-time PCR results of wound tissue showed that the levels of inflammatory cytokines il-1,il-6,TNF-radiation,il-17 and chemokines ip-10,il-8 and McP-1 in the wound tissue of diabetic foot ulcer rat model were significantly higher than that of normal ulcer model,and the levels of growth factor TGF-exposure were lower than that of normal ulcer model.LY-411575 significantly reduced il-1,il-6,TNF-maxima,il-17,and the chemokines ip-10,il-8,and McP-1 in diabetic foot ulcer rats,and reduced the expression of TGF-,TGF-earth.Jagged1 can increase the expression of TGF--,TGF---,suggesting that inhibition of the Notch signaling pathway can reduce the expression of the inflammatory factors il-1,il-6,TNF--,il-17a,il-8,and the growth factors TGF--,TGF---.Zizhu ointment can reduce the levels of il-1,il-6,TNF-benand,il-17,and the chemokines ip-10,il-8,and McP-1 on the wound surface of diabetic foot rats,and improve the expression of TGF-benand TGF-SUNS.Ly-411575 inhibited the expression of TGF-bento and TGF-promoting of Zizhu ointment.Conclusion:the expression of inflammatory cytokines and chemokines was higher and the expression of growth factors was lower in diabetic foot ulcer rats than in normal ulcer rats.Inhibition of Notch signaling pathway can reduce the expression of inflammatory factors,chemokines and growth factors in experimental model rats,and Notch signaling pathway can promote inflammation and cell proliferation.Zizhu ointment can reduce the levels of inflammatory cytokines and chemokines in diabetic foot ulcer rats,improve the expression of growth factors,and reduce wound inflammation,which may be related to the inhibition of Nocth signal expression.

Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day （E） 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

Transforming growth factor-β and fibrosis

Transforming growth factor-β （TGF-β）, a prototype of multifunctional cytokine, is a key regulator of extracellular matrix （ECM） assembly and remodeling. Specifically, TGF-β isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Elevated TGF-β expression in affected organs, and subsequent deregulation of TGF-β functions, correlates with the abnormal connective tissue deposition observed during the onset of fibrotic diseases. During the last few years, tremendous progress has been made in the understanding of the molecular aspects of intracellular signaling downstream of the TGF-β receptors. In particular, Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. The role of Smad3 in the transcriptional regulation of type I collagen gene expression and in the development of fibrosis, demonstrated both/n vitro and in animal models with a targeted deletion of Smad3, is of critical importance because it may lead to novel therapeutic strategies against these diseases. This review focuses on the mechanisms underlying Smad modulation of fibrillar collagen expression and how it relates to fibrotic processes.

TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

Objective To determine whether transforming growth factor betal (TGF-β1)/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines.Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study.TGF-β1-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay.For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements.After transfection, cells were treated with TGF-β1, then assayed for luciferase activity.Results The apoptosis rate of HepG2 cell lines (48.51%± 8.21%) was significantly higher than control ( 12.72%±2.18%, P<0.05).But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines.The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4.38) was significantly higher than control (1.00, P< 0.05).But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control.Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines.Smad4 is a central mediator of TGF-β1 signaling transdution pathway.TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Regulatory Effect of Shenge Yifei Capsule on TGF-β1/Smad Signaling Pathway in Rats with Chronic Obstructive Pulmonary Disease

[Objectives]This study aimed to study the effects of Shenge Yifei capsule on the TGF-β1/Smad signaling pathway in rats with chronic obstructive pulmonary disease(COPD).[Methods]Ten rats were randomly selected as the control group,and the other 40 rats were selected for modeling by fumigation combined with Klebsiella pneumoniae infection.A total of 38 rats were successfully modeled.They were randomly divided into model group(8 rats),low-dose Shenge Yifei capsule group(10 rats),high-dose Shenge Yifei capsule group(10 rats)and theophylline group(10 rats)in accordance with the principle of half male and half female.The rats in the model and control groups were given with distilled water by gavage,and the rats in the drug administration groups were given with corresponding drugs.The TGF-β1 level in the serum,and the expression levels of TGF-β1,Smad2,Smad3 and Smad7 and TGF-β1,Smad3 and Smad7 in airway tissues were detected.[Results]After 12 weeks,the serum TGF-β1 levels of the theophylline group and high-dose Shenge Yifei capsule group were lower than that of the low-dose Shenge Yifei capsule group(P<0.05).The expression levels of TGF-β1 and Smad3 in the theophylline group and high-dose Shenge Yifei capsule group were lower than that in the low-dose Shenge Yifei capsule group(P<0.05).The expression levels of TGF-β1 and Smad3 in the high-dose Shenge Yifei capsule group were lower than those in the low-dose Shenge Yifei capsule group and theophylline group(P<0.05).The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups(P<0.05).The expression levels of Smad7 in the theophylline group and high-dose Shenge Yifei capsule group were higher than that in the low-dose Shenge Yifei capsule group(P<0.05).After 18 weeks,no significant difference was found in serum TGF-β1 level among the theophylline group and low and high-dose Shenge Yifei capsule groups(P>0.05).The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups.The expression level of Smad7 in the high-dose Shenge Yifei capsule group was lower than that in the theophylline group(P<0.05).[Conclusions]Shenge Yifei capsule can regulate the TGF-β1/Smads signaling pathway.They can down-regulate the expression of TGF-β1,Smad2 and Smad3 and up-regulate the expression of Smad7,reducing the degree of airway modeling,delaying the development of COPD disease.Conventional high-dose Shenge Yifei capsule is more effective in inhibiting the expression of Smad2.

Activation of phospholipase D activity in transforming growth factor-beta-induced cell growth inhibition

Cells regulate phospholipase D (PLD) activity in response to numerous extracellular signals. Here, we investigated the involvement of PLD activity in transforming growth factor-β(TGF-β1)-mediated growth inhibition of epithelial cells. TGFβ1 inhibits the growth of MDCK, Mv1Lu, and A-549 cells. In the presence of 0.4 % butanol, TGF-β1 induces an increase in the formation of phosp hat idylbutanol, a unique product catalyzed by PLD. TGF-β1 also induces an increase in phosphatidic acid (PA) level in A-549 and MDCK cells. TGF-β1 induces an increase in the levels of DAG labeled with [~3H]-myristic acid in A-549 and MDCK cells but not in Mv1Lu cells- No increase of DAG was observed in cells prelabeled with [~3H]-arachidonic acid.The data presented suggest that PLD activation is involved in the TGF-β1-induced cell growth inhibition.

Termination of TGF-β Superfamily Signaling Through SMAD Dephosphorylation——A Functional Genomic View

The transforming growth factor-β （TGF-β） and related growth factors activate a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. They play key roles in the pathogenesis of many diseases especially cancer, fibrotic diseases, autoimmune diseases and cardiovascular diseases. TGF-β receptor-mediated phosphorylation of R-SMADs represents the most critical step in the TGF-β signaling pathways that triggers a cascade of intracellular events from SMAD complex assembly in the cytoplasm to transcriptional control in the nucleus. Conversely, dephosphorylafion of R-SMADs is a key mechanism for terminating TGF-β signaling. Our labs have recently taken an integrated approach combining functional genomics, biochemistry and development biology to describe the isolation and functional characterization of protein phosphatase PPM1A in controlling TGF-β signaling. This article briefly reviews how dynamic phosphorylation and dephosphorylation of SMADs control or fine-tune the signaling strength and duration and ultimately the physiological consequences in TGF-β signaling.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-SMA, collagen I, fibronectin, and p-Smad2/3. CONCLUSIONS: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.

有氧运动调节转化生长因子β/Smad通路缓解db/db糖尿病小鼠的肝脏纤维化

背景:有氧运动可抑制糖尿病小鼠肝纤维化,但具体作用机制尚未阐明。目的:基于转化生长因子β/Smad信号通路探讨有氧运动改善db/db小鼠肝纤维化的作用机制。方法:将8周龄雄性db/db小鼠和年龄匹配的m/m小鼠随机分为m/m对照组、m/m+运动组、db/db对照组、db/db+运动组,每组10只,运动组小鼠接受12周有氧运动。运动结束后检测各组小鼠空腹血糖,进行葡萄糖耐量测试和胰岛素耐量测试;摘取肝脏计算肝脏指数,摘眼球取血检测生化指标;苏木精-伊红染色、油红O染色和Masson染色观察小鼠肝组织的病理变化,免疫组化检测肝组织中转化生长因子β1、p-Smad3的表达;Western blot检测肝组织中转化生长因子β1、Smad3、p-Smad3、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平。结果与结论:①与m/m组和m/m+运动组小鼠相比,db/db组小鼠体质量、肝质量、肝脏指数、空腹血糖、三酰甘油、总胆固醇、低密度脂蛋白和肌酶水平均显著升高(P<0.01);高密度脂蛋白水平显著降低(P<0.01);转化生长因子β1、p-Smad3、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平显著升高(P<0.01);葡萄糖、胰岛素耐量测试的曲线下面积显著增加(P<0.01);肝组织病理染色显示大量炎症细胞浸润,脂滴增多,明显纤维化;②与db/db组相比,db/db+运动组小鼠体质量、肝质量、肝脏指数、空腹血糖、三酰甘油、总胆固醇、低密度脂蛋白和肌酶水平显著降低(P<0.05或P<0.01);高密度脂蛋白水平显著升高(P<0.05);转化生长因子β1、p-Smad3、α-平滑肌肌动蛋白、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达显著降低(P<0.05或P<0.01);此外,糖耐量、胰岛素耐量测试的曲线下面积明显减少(P<0.01,P<0.05),肝脏病理损伤得到明显改善;③结果表明,有氧运动有效减轻了糖尿病小鼠肝纤维化,其机制可能与调控转化生长因子β/Smad信号通路有关。

Smad7 dependent expression signature highlights BMP2 and HK2 signaling in HSC transdifferentiation

AIM: To analyse the influence of Smad7, antagonist of transforming growth factor (TGF)-β canonical signaling pathways on hepatic stellate cell (HSC) transdifferentia-tion in detail. METHODS: We systematically analysed genes regulated by TGF-β/Smad7 in activated HSCs by microarray analy-sis and validated the results using real time polymerase chain reaction and Western blotting analysis. RESULTS: We identif ied 100 known and unknown tar-gets underlying the regulation of Smad7 expression and delineated 8 gene ontology groups. Hk2, involved in glycolysis, was one of the most downregulated proteins, while BMP2, activator of the Smad1/5/8 pathway, was extremely upregulated by Smad7. However, BMP2 de-pendent Smad1 activation could be inhibited in vitro by Smad7 overexpression in HSCs. CONCLUSION: We conclude (1) the existence of a tight crosstalk of TGF-β and BMP2 pathways in HSCs and (2) a Smad7 dependently decreased sugar metabolism ameliorates HSC activation probably by energy with-drawal.

D-tryptophan triggered epithelial-mesenchymal transition by activating TGF-βsignaling pathway

D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to investigate D-tryptophan physiological function and demonstrate the underlying mechanisms.D-tryptophan suppressed HaCaT cell proliferation but increased cell migration.Specifically,D-tryptophan decreased E-cadherin and increased Snail,Twist,and Slug expression,resulting in the development of an epithelial-mesenchymal transitions(EMT)phenomenon.Moreover,D-tryptophan promoted the expression of transforming growth factor-β(TGF-β)1,and Smad4 knockout damages D-tryptophan’s ability.These results indicated that D-tryptophan stimulated HaCaT cells to produce TGF-β1 and thus activated the TGF-β/Samd pathway,resulting in the triggering of EMT.This study revealed the molecular mechanisms of D-tryptophan activity,provided D-tryptophan as a potential approach for cancer treatment,wound healing,organ development and other relevant applications.

Smad3 phospho-isoform signaling in hepatitis C virus-related chronic liver diseases

The risk of hepatocellular carcinoma (HCC) development increases as hepatitis virus C (HCV)-related liver diseases progress, especially in patients with active inflammation. Insight into hepatic carcinogenesis have emerged from recent detailed analyses of transforming growth factor-&#x003b2; and c-Jun-N-terminal kinase signaling processes directed by multiple phosphorylated (phospho)-isoforms of a Smad3 mediator. In the course of HCV-related chronic liver diseases, chronic inflammation and host genetic/epigenetic alterations additively shift the hepatocytic Smad3 phospho-isoform signaling from tumor suppression to carcinogenesis, increasing the risk of HCC. Chronic inflammation represents an early carcinogenic step that provides a nonmutagenic tumor-promoting stimulus. After undergoing successful antiviral therapy, patients with chronic hepatitis C could experience a lower risk of HCC as Smad3 phospho-isoform signaling reverses from potential carcinogenesis to tumor suppression. Even after HCV clearance, however, patients with cirrhosis could still develop HCC because of sustained, intense carcinogenic Smad3 phospho-isoform signaling that is possibly caused by genetic or epigenetic alterations. Smad3 phospho-isoforms should assist with evaluating the effectiveness of interventions aimed at reducing human HCC.