RESUMO
BACKGROUND: To date, the difference in microRNA expression profiles in tears of dry eye patients and healthy people has not been reported. In current study, we evaluated the significance of microRNAs and transforming growth factor beta2 (TGF-ß2) in distinguishing dry eye. METHODS: A total of 138 patients with dry eye from October 2017 to October 2018 were selected. During the same period, 138 healthy persons were collected. All patients were followed up for 12 months through outpatient, telephone or medical records and the time of corneal injury was recorded. RESULTS: Compared with healthy people, TGF-ß2 concentrations in dry eye patients were significantly decreased (P < 0.05). Array analysis, predictive software and dual-luciferase reporter assays showed that miR-450b-5p, miR-1283 and miR-3671 can target TGF-ß2 expression. Tear miR-450b-5p, miR-1283 and miR-3671 concentrations were significantly higher in dry eye patients than healthy people. A logistic regression model combining miR-450b-5p, miR-1283, miR-3671 and TGF-ß2 was performed. This model presented a high discriminating value (AUC: 0.907, 0.876-0.939, P < 0.001) than any single indicator, and the sensitivity and specificity were 77.7% and 92.7%, respectively. Compared with the low miR-450b-5p, low miR-1283, low miR-3671 and high TGF-ß2 groups, the high miR-450b-5p, high miR-1283, high miR-3671 and low TGF-ß2 groups had a significantly higher probability of corneal injury (TGF-ß2: χ2 = 5.762, P = 0.016; miR-450b-5p: χ2 = 13.267, P < 0.001; miR-1283: χ2 = 19.431, P < 0.001; miR-3671: χ2 = 8.131, P = 0.004). CONCLUSION: Current model combining tear miR-450b-5p, miR-1283, miR-3671 and TGF-ß2 had important values in the identification of dry eye and was of great value in evaluating the risk of corneal injury.
Assuntos
Síndromes do Olho Seco/metabolismo , Regulação da Expressão Gênica , MicroRNAs/biossíntese , Lágrimas/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Adulto , Idoso , Síndromes do Olho Seco/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiopoietina-1/biossíntese , Angiopoietina-1/genética , Linhagem Celular , Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/biossíntese , Cadeias beta de Integrinas/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/biossíntese , Fator de Crescimento Transformador beta3/genéticaRESUMO
Increasing studies have demonstrated that microRNAs (miRNAs) are associated with the metastasis of gallbladder carcinoma (GBC). Recently, miR-324-5p has been reported to be a tumour-suppressive miRNA in many types of malignant cancer. However, the biological function and molecular mechanism of miR-324-5p in GBC still remain largely unknown. Here, we found that miR-324-5p expression was notably down-regulated in both GBC tissues and cells compared with that in normal controls. Downregulated miR-324-5p expression was negatively associated with the status of local invasion and lymph node metastasis and predicted a poor prognosis in GBC patients. Further functional assays revealed that restoration of miR-324-5p significantly suppressed GBC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and impeded the metastasis of GBC cells in vivo. Moreover, RNA immunoprecipitation (RIP) and dual-luciferase reporter assay confirmed that the transforming growth factor beta 2 (TGFB2) was a direct target gene of miR-324-5p in GBC cells. Mechanically, small interfering RNA (siRNA)-mediated knockdown of TGFB2 partially phenocopied the inhibitory effects of miR-324-5p overexpression on GBC cell metastatic phenotypes. In summary, our findings demonstrated that miR-324-5p targets TGFB2 expression to inhibit GBC cell metastatic behaviors, and implying miR-324-5p as a potential biomarker for diagnostic and therapeutic strategies in GBC.
Assuntos
Regulação para Baixo , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Linhagem Celular Tumoral , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Humanos , MicroRNAs/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Fator de Crescimento Transformador beta2/genéticaRESUMO
Albeit most studies demonstrate the inhibitory role of G-quadruplex in the 5' Untranslated Region (5' UTR), our previous report depicted its completely contrasting activating role in the 5' UTR of transforming growth factor ß2 (TGFß2) mRNA. Therefore, we screened the 5' UTR of TGFß2 manually and identified a second putative G-quadruplex sequence. Our in vitro experiments encompassing CD and UV spectroscopy confirmed the ability of this sequence to form a G-quadruplex and in cellulo studies further indicated its activating role in modulation of TGFß2 gene expression. Our study suggests that these two 5' UTR G-quadruplexes most probably operate additively to substantially increase gene expression of TGFß2. Neither of the two G-quadruplex alone is sufficient enough to drastically augment protein production. Both G-quadruplexes are essential for increasing protein output. To the best of our knowledge, our study is the first report showcasing the combinatorial role of two G-quadruplexes in the 5' UTR of an mRNA.
Assuntos
Regiões 5' não Traduzidas , Quadruplex G , Regulação da Expressão Gênica , Fator de Crescimento Transformador beta2 , Humanos , Células MCF-7 , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genéticaRESUMO
Murine studies have linked TGF-ß signaling to emphysema, and human genome-wide association studies (GWAS) studies of lung function and COPD have identified associated regions near genes in the TGF-ß superfamily. However, the functional regulatory mechanisms at these loci have not been identified. We performed the largest GWAS of emphysema patterns to date, identifying 10 GWAS loci including an association peak spanning a 200 kb region downstream from TGFB2. Integrative analysis of publicly available eQTL, DNaseI, and chromatin conformation data identified a putative functional variant, rs1690789, that may regulate TGFB2 expression in human fibroblasts. Using chromatin conformation capture, we confirmed that the region containing rs1690789 contacts the TGFB2 promoter in fibroblasts, and CRISPR/Cas-9 targeted deletion of a ~ 100 bp region containing rs1690789 resulted in decreased TGFB2 expression in primary human lung fibroblasts. These data provide novel mechanistic evidence linking genetic variation affecting the TGF-ß pathway to emphysema in humans.
Assuntos
Enfisema/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Loci Gênicos , Predisposição Genética para Doença , Pulmão/patologia , Fator de Crescimento Transformador beta2/biossíntese , Idoso , Idoso de 80 Anos ou mais , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta2/genética , Estados UnidosRESUMO
BACKGROUND: Tendons are a crucial component of the musculoskeletal system and responsible for transmission forces derived from muscle to bone. Patients with tendon injuries are often observed with decreased collagen production and matrix degeneration, and healing of tendon injuries remains a challenge as a result of limited understanding of tendon biology. Recent studies highlight the contribution of miR-378a on the regulation gene expression during tendon differentiation. METHODS: We examined the tendon microstructure and tendon repair with using miR-378a knock-in transgenic mice, and the tendon-derived stem cells were also isolated from transgenic mice to study their tenogenic differentiation ability. Meanwhile, the expression levels of tenogenic markers were also examined in mouse tendon-derived stem cells transfected with miR-378a mimics during tenogenic differentiation. With using online prediction software and luciferase reporter assay, the binding target of miR-378a was also studied. RESULTS: Our results indicated miR-378a impairs tenogenic differentiation and tendon repair by inhibition collagen and extracellular matrix production both in vitro and in vivo. We also demonstrated that miR-378a exert its inhibitory role during tenogenic differentiation through binding at TGFß2 by luciferase reporter assay and western blot. CONCLUSIONS: Our investigation suggests that miR-378a could be considered as a new potential biomarker for tendon injury diagnosis or drug target for a possible therapeutic approach in future clinical practice.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Traumatismos dos Tendões/metabolismo , Tendões/metabolismo , Fator de Crescimento Transformador beta2/biossíntese , Animais , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Camundongos , Traumatismos dos Tendões/patologia , Tendões/patologiaRESUMO
Colorectal cancer patients often relapse after chemotherapy, owing to the survival of stem or progenitor cells referred to as cancer stem cells (CSCs). Although tumor stromal factors are known to contribute to chemoresistance, it remains not fully understood how CSCs in the hypoxic tumor microenvironment escape the chemotherapy. Here, we report that hypoxia-inducible factor (HIF-1α) and cancer-associated fibroblasts (CAFs)-secreted TGF-ß2 converge to activate the expression of hedgehog transcription factor GLI2 in CSCs, resulting in increased stemness/dedifferentiation and intrinsic resistance to chemotherapy. Genetic or small-molecule inhibitor-based ablation of HIF-1α/TGF-ß2-mediated GLI2 signaling effectively reversed the chemoresistance caused by the tumor microenvironment. Importantly, high expression levels of HIF-1α/TGF-ß2/GLI2 correlated robustly with the patient relapse following chemotherapy, highlighting a potential biomarker and therapeutic target for chemoresistance in colorectal cancer. Our study thus uncovers a molecular mechanism by which hypoxic colorectal tumor microenvironment promotes cancer cell stemness and resistance to chemotherapy and suggests a potentially targeted treatment approach to mitigating chemoresistance.
Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares/biossíntese , Fator de Crescimento Transformador beta2/biossíntese , Microambiente Tumoral , Proteína Gli2 com Dedos de Zinco/biossíntese , Hipóxia Celular , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fator de Crescimento Transformador beta2/genética , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
The aim of this study was to explore how atrial natriuretic polypeptide (ANP) affects the properties and function of endothelial cells. Gene expression data GSE56976 generated at 0, 1, and 6 hours after ANP incubation in human umbilical vein endothelial cells (HUVEC) was used. Microarray data were preprocessed for differentially expressed genes (DEGs) in each time-dependent group. Next, gene ontology (GO), pathway analysis, and transcriptional regulation were performed. Co-expression clustering analysis of DEGs and functional enrichment analysis of co-expression modules were processed. RT-PCR analysis was performed to validate gene expression. DEGs were obtained and their counts were increased from 0 hours to 6 hours. No overlapping DEGs were obtained among the 3 groups. The DEGs of ANP_6hours, including TGFB2 (transforming growth factor, beta 2), LTF (lactotransferrin/lactoferrin), and ETV7 (Ets variant 7) were mainly related with cell apoptosis and immune responses. The DEGs in the network of ANP_0hour were mainly associated with epithelial ion transport processes. In addition, 3 co-expressed modules were detected. CSF2 (colony stimulating factor 2) and PF4 (platelet factor 4) of the blue module were related with cytolysis, while FXYD1 (FXYD domain containing ion transport regulator 1) and TGFB2 of the yellow module were mainly enriched in ion transport and the ovulation cycle. The expression of TGFB2 obtained by microarray analysis was consistent with that of RT-PCR. Ion transport could be affected promptly after ANP treatment, and subsequently, the cytolysis of vein endothelial cells may be promoted and endothelial permeability would be enhanced, followed by activated immune responses.
Assuntos
Apoptose , Fator Natriurético Atrial/farmacologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Lactoferrina/genética , Proteínas Proto-Oncogênicas c-ets/genética , Fator de Crescimento Transformador beta2/genética , Células Cultivadas , Perfilação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Lactoferrina/biossíntese , Proteínas Proto-Oncogênicas c-ets/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta2/biossínteseRESUMO
End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-ß1, -ß2 and -ß3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-ß1, -ß2 and -ß3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-ß1, -ß2 and -ß3 among the groups (P > 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.
Assuntos
Anastomose Cirúrgica/efeitos adversos , Colágeno/metabolismo , Ducto Colédoco/patologia , Ducto Colédoco/cirurgia , Tamoxifeno/uso terapêutico , Fator de Crescimento Transformador beta/biossíntese , Animais , Ducto Colédoco/lesões , Ducto Colédoco/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fibrose , Masculino , RNA Mensageiro/genética , Sus scrofa , Tamoxifeno/farmacologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/biossíntese , Fator de Crescimento Transformador beta3/genética , Cicatrização/efeitos dos fármacosRESUMO
The importance of microRNA (miRNA) to vascular biology is becoming increasingly evident; however, the function of a significant number of miRNA remains to be determined. In particular, the effect of growth factor regulation of miRNAs on endothelial cell morphogenesis is incomplete. Thus, we aimed to identify miRNAs regulated by pro-angiogenic vascular endothelial growth factor (VEGF) and determine the effects of VEGF-regulated miRNAs and their targets on processes important for angiogenesis. Human umbilical vein endothelial cells (HUVECs) were thus stimulated with VEGF and miRNA levels assessed using microarrays. We found that VEGF altered expression of many miRNA, and for this study focused on one of the most significantly down-regulated miRNA in HUVECs following VEGF treatment, miR-30b. Using specific miRNA mimics, we found that overexpression of miR-30b inhibited capillary morphogenesis in vitro, while depletion of endogenous miR-30b resulted in increased capillary morphogenesis indicating the potential significance of down-regulation of miR-30b as a pro-angiogenic response to VEGF stimulation. MiR-30b overexpression in HUVEC regulated transforming growth factor beta 2 (TGFß2) production, which led to increased phosphorylation of Smad2, indicating activation of an autocrine TGFß signaling pathway. Up-regulation of TGFß2 by miR-30b overexpression was found to be dependent on ATF2 activation, a transcription factor known to regulate TGFß2 expression, as miR-30b overexpressing cells exhibited increased levels of phosphorylated ATF2 and depletion of ATF2 inhibited miR-30b-induced TGFß2 expression. However, miR-30b effects on ATF2 were indirect and found to be via targeting of the known ATF2 repressor protein JDP2 whose mRNA levels were indirectly correlated with miR-30b levels. Increased secretion of TGFß2 from HUVEC was shown to mediate the inhibitory effects of miR-30b on capillary morphogenesis as treatment with a neutralizing antibody to TGFß2 restored capillary morphogenesis to normal levels in miR-30b overexpressing cells. These results support that the regulation of miR-30b by VEGF in HUVEC is important for capillary morphogenesis, as increased miR-30b expression inhibits capillary morphogenesis through enhanced expression of TGFß2.
Assuntos
Capilares/crescimento & desenvolvimento , Endotélio Vascular/citologia , MicroRNAs/fisiologia , Fator de Crescimento Transformador beta2/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Morfogênese , Fosforilação , Fator de Crescimento Transformador beta2/biossíntese , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
PURPOSE: The fifth most common cancer is allocated to bladder cancer (BC) worldwide. Understanding the molecular mechanisms of BC invasion and metastasis to identify target therapeutic strategies will improve disease survival. So the aim of this study was to measure expression rate of zinc finger E-box binding homeobox 1 (ZEB1) and transforming growth factor-beta2 (TGF-ß2) mRNA in tissue samples of patients with BC and its healthy adjacent tissue samples and their association with muscle invasion, size and grade of the tumor. MATERIALS AND METHODS: Tissue samples were collected from 35 newly diagnosed untreated patients with BC from 2013 to 2014. Total RNA was extracted from about 50-mg tissue samples using TRIzol reagent. TAKARA SYBR Premix EX Tag II was applied to determine the rate of mRNA expression by real-time polymerase chain reaction (PCR). To obtain final validation, PCR product of ZEB1 and TGF-ß2 were sequenced. STATA 11 software was used to analyze the data. RESULTS: The expression level of ZEB1 in tumor samples was significantly more than of in healthy adjacent tissue samples. Up-regulation of TGF-ß2 showed a strong association with muscle invasion (p=0.017). There was also demonstrated a relationship between over expression of ZEB1 with the tumor size (p=0.050). CONCLUSIONS: It looks ZEB1 and TGF-ß2 had a role in BC patients. In this study ZEB1 expression was higher in BC tissues than that of in healthy control tissues. There was demonstrated a markedly association between overexpression of TGF-ß2 and muscle invasion. Therefore, they are supposed to be candidate as potential biomarkers for early detection and progression of BC.
Assuntos
Biomarcadores Tumorais/biossíntese , Fator de Crescimento Transformador beta2/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Neoplásico/genética , Manejo de Espécimes/métodos , Fator de Crescimento Transformador beta2/genética , Regulação para Cima , Neoplasias da Bexiga Urinária/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Purpose: The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFß2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFß2-TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods: Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFß2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 µM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFß2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results: Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFß2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFß2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFß2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions: These studies identify TGFß2-TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.
Assuntos
Humor Aquoso/metabolismo , Regulação da Expressão Gênica , Hipertensão Ocular/genética , RNA/genética , Receptor 4 Toll-Like/genética , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/genética , Animais , Western Blotting , Células Cultivadas , Humanos , Imuno-Histoquímica , Pressão Intraocular , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C3H , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Receptor 4 Toll-Like/biossíntese , Malha Trabecular/patologia , Fator de Crescimento Transformador beta2/biossínteseRESUMO
Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder with a range of cardiovascular, skeletal, craniofacial and cutaneous manifestations. LDS type 4 is caused by mutations in TGFß ligand 2 (TGFB2) and based on the family pedigrees described to date, appears to have a milder clinical phenotype, often presenting with isolated aortic disease. We sought to investigate its molecular basis in a new pedigree. We identified a missense variant p.(Arg320Cys) (NM_003238.3) in a highly evolutionary conserved region of TGFB2 in a new LDS type 4 pedigree with multiple cases of aortic aneurysms and dissections. There was striking upregulation of TGFB1 and TGFB2 expression on immunofluorescent staining, and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlights the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFß signaling in this emerging familial aortopathy.
Assuntos
Aneurisma Aórtico/genética , Síndrome de Loeys-Dietz/genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta2/genética , Aneurisma Aórtico/patologia , Regulação da Expressão Gênica , Humanos , Síndrome de Loeys-Dietz/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/biossínteseRESUMO
Transforming growth factor beta (TGF-ß) is an important cytokine with pleiotropic regulatory functions in the immune system and in the responses against viral infections. TGF-ß acts on a variety of immune cells through the cell surface TGF-ß receptor (University of Duisburg-EssenTGFBR). The woodchuck has been used as a biomedical model for studies of obesity and energy balance, endocrine and metabolic function, cardiovascular, cerebrovascular and neoplastic disease. Woodchucks infected with woodchuck hepatitis virus (WHV) represent an informative animal model to study hepatitis B virus (HBV) infection. In this study, the cDNA sequences of woodchuck TGF-ß1, TGF-ß2, TGFBR1 and TGFBR2 were cloned, sequenced and characterized. The full-length TGFBR1 cDNA sequence consisted of 1305bp coding sequence (CDS) that encoded 434 amino acids with a molecular weight of 48.9kDa. The phylogenetic tree analysis revealed that the woodchuck TGF-ß family genes had a closer genetic relationship with Ictidomys tridecemlineatus. One antibody with cross-reactivity to woodchuck TGFBR1 was identified by flow cytometry. Moreover, the expression of these genes were analyzed at the transcriptional level. The quantitative PCR analysis showed that the TGF-ß family transcripts were constitutively expressed in many tissues tested. Altered expression levels of the TGF-ß family transcripts in the liver of WHV infected woodchucks were observed. These results serve as a foundation for further insight into the role of the TGF-ß family in viral hepatitis in woodchuck model. Our work also possesses the potential value for characterizing the TGF-ß family in other related diseases, such as obesity-related diseases, metabolic disorder, cardiovascular disease and cancer.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta2 , Animais , Marmota/genética , Marmota/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genéticaRESUMO
PURPOSE: To investigate the progression of epiretinal membranes after intravitreal bevacizumab (IVB) injection therapy in patients with proliferative membranes and evaluate the changes in fibrosis-related cytokines in retinal pigment epithelial cells and glial cells after treatment with bevacizumab. METHODS: Retrospective study of the proliferative membranes in patients with and without IVB therapy. In vitro, the human adult retinal pigment epithelial (ARPE-19) cells and BV2 microglial cell lines were incubated in different bevacizumab concentrations under hypoxic conditions. Cell culture supernatants and cell lysates were harvested after incubation for 24, 48 or 72â hours for ELISA and western blot. RESULTS: Bevacizumab accelerated fibrosis in patients with proliferative membranes. Immunofluorescence analysis revealed more intense transforming growth factor ß2 (TGFß2) and connective tissue growth factor (CTGF) staining in IVB-treated proliferative diabetic retinopathy (PDR) membranes compared with membranes of patients not receiving IVB therapy. This result was consistent with real-time PCR results. Bevacizumab incubation significantly upregulated TGFß2 and CTGF in ARPE-19 cells and BV2 microglial cells, but ciliary neurotrophic factor (CNTF) expression was upregulated only in BV2 microglial cells. CONCLUSIONS: Anti-vascular endothelial growth factor treatment likely accelerates fibrosis in PDR patients via upregulation of TGFß2, CTGF and CNTF, suggesting the importance of adjunctive therapy for retinal fibrosis.
Assuntos
Bevacizumab/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Vitreorretinopatia Proliferativa/tratamento farmacológico , Corpo Vítreo/diagnóstico por imagem , Inibidores da Angiogênese/administração & dosagem , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrose , Seguimentos , Regulação da Expressão Gênica , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Tempo , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vitreorretinopatia Proliferativa/diagnóstico , Vitreorretinopatia Proliferativa/metabolismo , Corpo Vítreo/efeitos dos fármacosRESUMO
The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.
Assuntos
Fissura Palatina/embriologia , Fatores de Transcrição Forkhead/fisiologia , Mesoderma/embriologia , Palato/embriologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Animais , Colágeno/fisiologia , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrinas/fisiologia , Mandíbula/embriologia , Maxila/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoglicanas/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Língua/anormalidades , Língua/embriologia , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genéticaRESUMO
Pulmonary vascular endothelial function may be impaired by oxidative stress in endotoxemia-derived acute lung injury. Growing evidence suggests that endothelial-to-mesenchymal transition (EndMT) could play a pivotal role in various respiratory diseases; however, it remains unclear whether EndMT participates in the injury/repair process of septic acute lung injury. Here, we analyzed lipopolysaccharide (LPS)-treated mice whose total number of pulmonary vascular endothelial cells (PVECs) transiently decreased after production of reactive oxygen species (ROS), while the population of EndMT-PVECs significantly increased. NAD(P)H oxidase inhibition suppressed EndMT of PVECs. Most EndMT-PVECs derived from tissue-resident cells, not from bone marrow, as assessed by mice with chimeric bone marrow. Bromodeoxyuridine-incorporation assays revealed higher proliferation of capillary EndMT-PVECs. In addition, EndMT-PVECs strongly expressed c-kit and CD133. LPS loading to human lung microvascular endothelial cells (HMVEC-Ls) induced reversible EndMT, as evidenced by phenotypic recovery observed after removal of LPS. LPS-induced EndMT-HMVEC-Ls had increased vasculogenic ability, aldehyde dehydrogenase activity, and expression of drug resistance genes, which are also fundamental properties of progenitor cells. Taken together, our results demonstrate that LPS induces EndMT of tissue-resident PVECs during the early phase of acute lung injury, partly mediated by ROS, contributing to increased proliferation of PVECs.
Assuntos
Lesão Pulmonar Aguda/imunologia , Células Progenitoras Endoteliais/fisiologia , Lipopolissacarídeos/farmacologia , Lesão Pulmonar Aguda/patologia , Animais , Apoptose , Proliferação de Células , Transdiferenciação Celular , Células Cultivadas , Células Progenitoras Endoteliais/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Feminino , Expressão Gênica , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genéticaRESUMO
Transforming growth factor-beta (TGF-ß) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-ß2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-ß2 action in TNBC cells. Breast cancer patients with high TGF-ß2 expression have a poor prognosis. The levels of TGF-ß2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-ß receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-ß2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-ß2 mRNA expression level but not that of TGF-ß1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-ß2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Silimarina/farmacologia , Fator de Crescimento Transformador beta2/biossíntese , Neoplasias de Mama Triplo Negativas/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real , Silibina , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Prognóstico , Fator de Crescimento Transformador beta2/biossínteseRESUMO
Elevated intraocular pressure (IOP) is causally implicated in the pathophysiology of primary open-angle glaucoma (POAG). The molecular mechanisms responsible for elevated IOP remain elusive, but may involve aberrant expression and signaling of transforming growth factor (TGF)-ß2 within the trabecular meshwork (TM). Consistent with previously published studies, we show here that exogenous addition of TGF-ß2 to cultured porcine anterior segments significantly attenuates outflow facility in a time-dependent manner. By comparison, perfusing segments with a TGFßRI/ALK-5 antagonist (SB-431542) unexpectedly elicited a significant and sustained increase in outflow facility, implicating a role for TM-localized constitutive expression and release of TGF-ß2. Consistent with this thesis, cultured primary or transformed (GTM3) quiescent human TM cells were found to constitutively express and secrete measurable amounts of biologically-active TGF-ß2. Disrupting monomeric GTPase post-translational prenylation and activation with lovastatin or GGTI-298 markedly reduced constitutive TGF-ß2 expression and release. Specifically, inhibiting the Rho subfamily of GTPases with C3 exoenzyme similarly reduced constitutive expression and secretion of TGF-ß2. These findings suggest that Rho GTPase signaling, in part, regulates constitutive expression and release of biologically-active TGF-ß2 from human TM cells. Localized constitutive expression and release of TGF-ß2 by TM cells may promote or exacerbate elevation of IOP in POAG.