Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells

BACKGROUND Hepatic stellate cells(HSCs)are the key effector cells mediating the occurrence and development of liver fibrosis,while aerobic glycolysis is an important metabolic characteristic of HSC activation.Transforming growth factor-β1(TGF-β1)induces aerobic glycolysis and is a driving factor for metabolic reprogramming.The occurrence of glycolysis depends on a high glucose uptake level.Glucose transporter 1(GLUT1)is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism,thus affecting cell proliferation and growth.However,little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIM To investigate the mechanisms of action of GLUT1,TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue.A Seahorse extracellular flux(XF)analyzer was used to examine changes in aerobic glycolytic flux,lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation.The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothersagainst-decapentaplegic-homolog 2/3(Smad2/3)signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase(PI3K)/AKT signaling pathways.In addition,GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs.Finally,a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues.In addition,immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins,indicating that GLUT1 expression was related to the development of liver fibrosis.TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad,p38 MAPK and P13K/AKT signaling pathways.The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression.GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration.A GLUT1 inhibitor was administered in a mouse model of liver fibrosis,and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSION TGF-β1 induces GLUT1 expression in HSCs,a process related to liver fibrosis progression.In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs.In addition,in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

基于生物信息学分析TGFBI基因在头颈部鳞癌中的表达及其预后意义

目的探讨转化生长因子β诱导基因(transforming growth factorβ-induced gene,TGFBI)在头颈部鳞癌(head and neck squamous carcinoma,HNSC)中的表达及其预后意义。方法首先通过TIMER2.0数据库分析TGFBI基因在泛癌中的表达水平;并进一步利用UALCAN数据库和GEPIA数据库检索TGFBI基因在HNSC与正常组织中的表达水平,以及TGFBI基因与患者种族、年龄、性别、肿瘤分期、淋巴结转移状况等相关临床特征的关系;通过STRING数据库分析TGFBI基因的互作蛋白,借助DAVID数据库对其与互作蛋白基因进行功能富集分析;最后通过GEPIA数据库分析TGFBI基因在HNSC中的生存意义。结果发现TGFBI在HNSC中的表达明显高于正常组织(P<0.05),TGFBI高表达的患者总生存期明显低于低表达的患者(P<0.05)。蛋白互作分析发现,FN1、MMP14、ITGA3、ITGAM、ITGAV、ITGB1、ITGB2、ITGB3、TGIF2和TGIF2LX这10个蛋白基因与TGFBI存在紧密联系,此外,TGFBI及相关基因功能富集分析显示它们主要参与了5个生物学程序、4个细胞组分、3个分子功能、4个信号通路。结论TGFBI在HNSC中的表达水平较正常组织明显升高,且与患者预后不良有关,有望成为HNSC诊断和治疗的生物学靶点。

Association of Chinese Medicine Constitution Susceptibility to Diabetic Nephropathy and Transforming Growth Factor-β1(T869C)Gene Polymorphism

Objective：To explore the association of Chinese medicine constitution susceptibility to diabetic nephropathy（DN） and transforming growth factor（TGF）-β1（T869C） gene polymorphism.Methods：TGF-β1 gene polymorphism detected with polymerase chain reaction-restriction fragment length polymorphism（PCRRFLP） was screened for 180 DN cases and 180 type 2 diabetic mellitus（T2DM） cases without combined DN. Patients with DN were surveyed epidemiologically with constitution in the Chinese medicine questionnaire （CCMQ）.Binary logistic regression analysis was utilized to study the correlation between nine types of Chinese medicine constitution and TGF-β1（T869C） gene polymorphisms.Results：The DN group has a higher frequency of TGF-β1（T869C） gene polymorphism than the T2DM group,and CC/CT genotypes than the T2DM group[CC,CT,TT（DN group）：88,87,5（cases） versus（T2DM group） 71,73,36（cases）,P0.05].The phlegm-dampness constitution,damp-heat constitution,and blood stasis constitution have correlations with TGF-β1 （T869C） gene polymorphism.Conclusion：Chinese medicine constitutions were associated with TGF-β1（T869C） gene polymorphism,a potential predictor of susceptibility to DN in T2DM patients.

Inhibition of TGF-β1 by eNOS gene transfer provides cardiac protection after myocardial infarction

Objective： Endothelial nitric oxide synthase （eNOS） and nitric oxide （NO） have been implicated in protection against myocardial ischemia injury. This study was designed to explore a new method of therapy for myocardial injury by eNOS gene transfection. Methods： A rat model of myocardial infarction （MI） was established by left anterior descending （LAD） coronary artery ligation, eNOS gene in an adenovirus vector was delivered locally into the rat heart and hemodynamic parameters were examined after 3 weeks, Matrix metalloproteinase-2 and 9 （MMP-2, MMP-9） mRNA were measured by reverse transcription polymerase chain reaction （RT-PCR）, and the protein levels of eNOS, caspase-3, and transforming grouth factor 131 （TGF-131） were determined by western blot assay. Results： eNOS gene transfer significantly reduced cardiomyocyte apoptosis and improved cardiac function. In addition, eNOS significantly reduced the mRNA levels of MMP-2 and MMP-9. In the eNOS gene transfected group, the activation of caspase-3 and TGF-β1 were decreased. However, the protection was reversed by administration of the NOS inhibitor, N（o））-nitro-l-arginine methyl ester （L-NAME）. Conclusion： These results demonstrate that the eNOS provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis and collagen deposition, and suppression of TGF-β1.

海南省糖尿病视网膜病变患者TGFBI基因突变情况分析

目的调查分析转化生长因子β诱导基因(TGFBI)基因突变在海南省糖尿病视网膜病变患者中的发生情况。方法选择2013年8月至2018年2月在海南医学院第一附属医院眼科就诊的海南省糖尿病视网膜病变患者78例作为观察组,同期选择本省非糖尿病视网膜病变患者68例作为对照组,两组患者均进行全血组织TGFBI基因突变检测,记录患者的临床特征并进行相关性分析。结果观察组患者的TGFBI基因突变率为10.25%,明显高于对照组的1.47%,差异有统计学意义(P<0.05);8例TGFBI基因突变患者中,-51G>A 3例,-26A>G 3例,310G>A 1例,895G>T 1例;Pearson相关分析结果显示,海南省糖尿病视网膜病变患者的TGFBI基因突变与最佳矫正视力、视网膜厚度、眼压均呈正相关性(P<0.05)。结论TGFBI基因突变在海南省糖尿病视网膜病变患者中比较常见,与患者的临床特征显著相关。

Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.

转化生长因子β诱导的基因抑制恶性间皮瘤细胞增殖的体外研究

目的研究转化生长因子β诱导的基因(transforming growth factor-βinduced gene,TGFBI)在体外是否能抑制恶性间皮瘤细胞NCI-H28的增殖。方法将外源性TGFBI稳定性转染到恶性间皮瘤细胞NCI-H28中,绘制生长曲线图,测定其细胞增殖、接种效率和软琼脂克隆形成,并进行细胞周期的分析。结果外源性TGFBI在恶性间皮瘤细胞NCI-H28中能够稳定地高表达;当外源性的TGFBI转入到肿瘤细胞NCI-H28后,TGFBI高表达的细胞倍增时间增加了4.38倍;与转染空载体的肿瘤细胞相比:NCI-H28的相对接种效率降低了69.68%,外源性TGFBI表达的T28细胞形成了较小的软琼脂克隆;TGFBI可以使肿瘤细胞阻滞在G1期,并延缓肿瘤细胞进入S期的时间。结论 TGFBI可以抑制恶性间皮瘤细胞的体外增殖。

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.

Pathogenic mutations of TGFBI and CHST6 genes in Chinese patients with Avellino,lattice,and macular corneal dystrophies

Objective:To investigate gene mutations associated with three different types of corneal dystrophies(CDs),and to establish a phenotype-genotype correlation.Methods:Two patients with Avellino corneal dystrophy(ACD),four patients with lattice corneal dystrophy type I(LCD I) from one family,and three patients with macular corneal dystrophy type I(MCD I) were subjected to both clinical and genetic examinations.Slit lamp examination was performed for all the subjects to assess their corneal phenotypes.Genomic DNA was extracted from peripheral blood leukocytes.The coding regions of the human transforming growth factor β-induced(TGFBI) gene and carbohydrate sulfotransferase 6(CHST6) gene were amplified by polymerase chain reaction(PCR) and subjected to direct sequencing.DNA samples from 50 healthy volunteers were used as controls.Results:Clinical examination showed three different phenotypes of CDs.Genetic examination identified that two ACD subjects were associated with homozygous R124H mutation of TGFBI,and four LCD I subjects were all associated with R124C heterozygous mutation.One MCD I subject was associated with a novel S51X homozygous mutation in CHST6,while the other two MCD I subjects harbored a previously reported W232X homozygous mutation.Conclusions:Our study highlights the prevalence of codon 124 mutations in the TGFBI gene among the Chinese ACD and LCD I patients.Moreover,we found a novel mutation among MCD I patients.

Emerging targets in cancer drug resistance

Drug resistance is a complex phenomenon that frequently develops as a failure to chemotherapy during cancer treatment.Malignant cells increasingly generate resistance to various chemotherapeutic drugs through distinct mechanisms and pathways.Understanding the molecular mechanisms involved in drug resistance remains an important area of research for identification of precise targets and drug discovery to improve therapeutic outcomes.This review highlights the role of some recent emerging targets and pathways which play critical role in driving drug resistance.