RESUMO
BACKGROUND: Although aberrant expression of CDGSH iron sulfur domain 2 (CISD2) contributes to the tumorigenesis and progression of numerous human cancers, the biological function of CISD2 and its specific prognostic value in lung squamous cell carcinoma (LUSC) have yet to be comprehensively explored. The current study aimed to elucidate the role of CISD2 in LUSC as well as the underlying molecular mechanisms. METHODS: Immunohistochemistry was conducted to detect the protein expression of CISD2 and analyze whether high expression of CISD2 affects the overall survival (OS) of LUSC patients. Cell proliferation, colony formation, wound healing and Transwell invasion assays were performed to clarify whether CISD2 contributes to LUSC cell proliferation and disease progression. Quantitative real-time reverse transcription-PCR and western blot assays were used to detect the levels of transcription factors and key epithelial-mesenchymal transition (EMT)-related markers in LUSC cells after CISD2 knockdown and overexpression to determine whether CISD2 regulates transforming growth factor-beta (TGF-ß)-induced EMT in LUSC. RESULTS: Immunohistochemistry of human tissue microarrays containing 90 pairs of adjacent and cancerous tissues revealed that CISD2 is considerably overexpressed in LUSC and strongly linked to poor OS. Functional experiments suggested that silencing endogenous CISD2 inhibited the growth, colony formation, migration, and invasion of H2170 and H226 cell lines. Exogenous overexpression of CISD2 facilitated these phenotypes in SK-MES-1 and H2170 cells. Furthermore, CISD2 promoted EMT progression by increasing the expression of mesenchymal markers (N-cadherin, vimentin, Snail, and Slug) as well as SMAD2/3 and reducing the expression of the epithelial marker E-cadherin. Mechanistically, our studies provide the first evidence that CISD2 can promote EMT by enhancing TGF-ß1-induced Smad2/3 expression in LUSC cells. CONCLUSION: In conclusion, our research illustrates that CISD2 is highly expressed in LUSC and may facilitate LUSC proliferation and metastasis. Thus, CISD2 may serve as an independent prognostic marker and possible treatment target for LUSC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/fisiologia , Pulmão , Neoplasias Pulmonares/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Müller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-ß (TGF-ß) induces Müller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing α-smooth muscle actin (α-SMA). Our aim was to investigate the effect of the TGF-ß inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Müller cells. MIO-M1 human Müller cells were treated with TGF-ß1 (10 ng/mL), galunisertib (5, 10 and 20 µM) and TGF-ß1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-ß1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and α-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Müller cells incorporated. Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Müller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-ß1 pathway by decreasing phospho-SMAD3 immunoreactivity, attenuated the α-SMA expression, and prevented the contraction of Müller cells in collagen gel. Although more studies are needed, in vitro assays suggest that galunisertib may be a potential candidate to attenuate the formation of fibrocontractile membranes and prevent retinal detachment and consequent loss of vision.
Assuntos
Células Ependimogliais , Membrana Epirretiniana , Humanos , Células Ependimogliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Neuroglia/metabolismo , Actinas/metabolismo , Colágeno/metabolismo , Membrana Epirretiniana/metabolismoRESUMO
Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-ß1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-ß1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-ß1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-ß, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-ß1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-ß1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-ß1 can reduce the adhesive properties of these cells; however, direct effects of TGF-ß1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed.
Assuntos
Anemia Falciforme , Neutrófilos , Fator de Crescimento Transformador beta1 , Doenças Vasculares , Anemia Falciforme/complicações , Anemia Falciforme/metabolismo , Animais , Humanos , Inflamação/metabolismo , Camundongos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Doenças Vasculares/metabolismoRESUMO
Octreotide (OCT) is used to inhibit hormone secretion and growth in somatotroph tumors, although a significant percentage of patients are resistant. It has also been tested in nonfunctioning (NF) tumors but with poor results, with these outcomes having been associated with SSTR2 levels and impaired signaling. We investigated whether OCT inhibitory effects can be improved by TGF-ß1 in functioning and nonfunctioning somatotroph tumor cells. OCT effects on hormone secretion and proliferation were analyzed in the presence of TGF-ß1 in WT and SSTR2-overexpressing secreting GH3 and silent somatotroph tumor cells. The mechanism underlying these effects was assessed by studying SSTR and TGFßR signaling pathways mediators. In addition, we analyzed the effects of OCT/TGF-ß1 treatment on tumor growth and cell proliferation in vivo. The inhibitory effects of OCT on GH- and PRL-secretion and proliferation were improved in the presence of TGF-ß1, as well as by SSTR2 overexpression. The OCT/TGF-ß1 treatment induced downregulation of pERK1/2 and pAkt, upregulation of pSmad3, and inhibition of cyclin D1. In vivo experiments showed that OCT in the presence of TGF-ß1 blocked tumor volume growth, decreased cell proliferation, and increased tumor necrosis. These results indicate that SSTR2 levels and the stimulation of TGF-ß1/TGFßR/Smad2/3 pathway are important for strengthening the antiproliferative and antisecretory effects of OCT.
Assuntos
Antineoplásicos Hormonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Octreotida/farmacologia , Neoplasias Hipofisárias/tratamento farmacológico , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Somatotrofos/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Linhagem Celular , Feminino , Humanos , Camundongos Nus , Fosforilação , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Ratos , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Transdução de Sinais , Somatotrofos/metabolismo , Somatotrofos/patologia , Carga Tumoral/efeitos dos fármacosRESUMO
Transforming growth factor-beta 1 (TGF-ß1) is a cytokine with marked pro-fibrotic action on cardiac fibroblasts (CF). TGF-ß1 induces CF-to-cardiac myofibroblast (CMF) differentiation, defined by an increase in α-smooth muscle cells (α-SMA), collagen secretion and it has a cytoprotective effect against stimuli that induce apoptosis. In the Endoplasmic Reticulum (ER) lumen, misfolded protein accumulation triggers ER stress and induces apoptosis, and this process plays a critical role in cell death mediated by Ischemia/Reperfusion (I/R) injury and by ER stress inducers, such as Tunicamycin (Tn). Here, we studied the regulation of CHOP, a proapoptotic ER-stress-related transcription factor in CF under simulated I/R (sI/R) or exposed to Tn. Even though TGF-ß1 has been shown to participate in ER stress, its regulatory effect on CF apoptosis and ER stress-induced by sI/R or TN has not been evaluated yet. CF from neonatal rats were exposed to sI/R, and cell death was evaluated by cell count and apoptosis by flow cytometry. ER stress was assessed by western blot against CHOP. Our results evidenced that sI/R (8/24) h or Tn triggers CF apoptosis and an increase in CHOP protein levels. TGF-ß1 pre-treatment partially prevented apoptosis induced by sI/R or Tn. Furthermore, TGF-ß1 pre-treatment completely prevented CHOP increase by sI/R or Tn. Additionally, we found a decrease in α-SMA expression induced by sI/R and in collagen secretion induced by Tn, which were not prevented by TGF-ß1 treatment. In conclusion, TGF-ß1 partially protects CF apoptosis induced by sI/R or Tn, through a mechanism that would involve ER stress.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/citologia , Ratos Sprague-Dawley , Tunicamicina/farmacologiaRESUMO
Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-ß on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-ß1 under hypoxic conditions. Hypoxia and TGF-ß1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-ß signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-ß1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-ß signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Esquelético/patologia , Fator de Crescimento Transformador beta1/administração & dosagem , Regulação para Cima , Animais , Diferenciação Celular , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Injeções Intramusculares , Isquemia/etiologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Células NIH 3T3 , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Follicular CD4+ T cells are the main HIV reservoirs due to, among other factors, the low frequency of CD8+ T cells in lymphoid follicles. Follicular CXCR5+ CD8+ T cells are associated with HIV control, but their differentiation conditions are yet undefined. In this study, we explored the in vitro effect of transforming growth factor (TGF)-ß1, interleukin (IL)-12, and IL-23 on the induction of CXCR5, the follicle homing receptor, in human circulating CD8+ T cells from seronegative, and treated HIV-infected individuals. The combination of TGF-ß1 plus IL-23 induced the highest expression of CXCR5 in purified CD8+ T cells. These CXCR5+ CD8+ T cells also expressed a transcriptional and phenotypic profile similar to that of follicular CD4+ T cells, such as the upregulation of BCL6, inducible costimulator and CD40L, and downregulation of PRDM1. These cells responded in vitro to CXCL13 and had low expression of CCR7. In addition, after polyclonal stimulation, they produced IL-21, interferon-γ, and de novo perforin. However, in comparison with seronegative individuals, CD8+ T cells from HIV-infected patients had a lower response to TGF-ß1/IL-23, a defect that was restored with the blockade of the programmed cell death 1 inhibitory receptor. Thus, TGF-ß1 plus IL-23 induce follicular-like CXCR5+ CD8+ T cells in seronegative individuals, but in HIV-infected patients there is a limited response which could impair the generation of this cell population.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Interleucina-23/farmacologia , Receptores CXCR5/imunologia , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Quimiocina CXCL13/farmacologia , Soronegatividade para HIV/imunologia , Humanos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Adulto JovemRESUMO
The aryl hydrocarbon receptor (AHR) is a transcription factor activated by ligand highly expressed on TH17 cells, and AHR-deficient CD4+ T cells have impaired production of IL-17A and IL-22. Although AHR activation can exacerbate in vivo TH17 cell-mediated autoimmunity, accumulating data indicate that AHR is a nonpathogenic TH17 marker. Thus it remains unclear how AHR activation is regulated and impacts on the generation of TH17 subsets. Here we demonstrated that AHR pathway is activated during in vitro pathogenic TH17 polarization, but it is quickly downregulated. Under these conditions, additional AHR activation promoted IL-22 but not IL-17A. Interestingly, AHR high sustained expression and IL-17A promotion were only achieved when TGFß1 was present in the culture. In addition to the effect on AHR regulation, TGFß1 presented a dual role by simultaneously suppressing the TH17 pathogenic phenotype acquisition. This latter effect was independent of AHR stimulation, since its activation did not confer a TH17 anti-inflammatory profile and Ahr-/- cells did not upregulate any TH17 pathogenic marker. Through the use of EAE model, we demonstrated that AHR is still functional in encephalitogenic CD4+ T cells and the adoptive transfer of Ahr-/- TH17 cells to recipient mice resulted in milder EAE development when compared to their WT counterparts. Altogether, our data demonstrated that although AHR is highly expressed on in vitro-generated nonpathogenic TH17 cells, its ligation does not shift TH17 cells to an anti-inflammatory phenotype. Further studies investigating the role of AHR beyond TH17 differentiation may provide a useful understanding of the physiopathology of autoimmune diseases.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta1/farmacologia , Transferência Adotiva , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-17/genética , Interleucina-17/imunologia , Interleucinas/genética , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fenótipo , Cultura Primária de Células , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Células Th17/efeitos dos fármacos , Células Th17/patologia , Células Th17/transplante , Interleucina 22RESUMO
Transforming growth factor-ß1 (TGF-ß1) is a potent inducer of fibroblast to myofibroblast differentiation and contributes to the pro-fibrotic microenvironment during cardiac remodeling. Fibroblast growth factor-2 (FGF-2) is a growth factor secreted by adipose tissue-derived stromal cells (ASC) which can antagonize TGF-ß1 signaling. We hypothesized that TGF-ß1-induced cardiac fibroblast to myofibroblast differentiation is abrogated by FGF-2 and ASC conditioned medium (ASC-CMed). Our experiments demonstrated that TGF-ß1 treatment-induced cardiac fibroblast differentiation into myofibroblasts, as evidenced by the formation of contractile stress fibers rich in αSMA. FGF-2 blocked the differentiation, as evidenced by the reduction in gene (TAGLN, p < 0.0001; ACTA2, p = 0.0056) and protein (αSMA, p = 0.0338) expression of mesenchymal markers and extracellular matrix components gene expression (COL1A1, p < 0.0001; COL3A1, p = 0.0029). ASC-CMed did not block myofibroblast differentiation. The treatment with FGF-2 increased matrix metalloproteinases gene expression (MMP1, p < 0.0001; MMP14, p = 0.0027) and decreased the expression of tissue inhibitor of metalloproteinase gene TIMP2 (p = 0.0023). ASC-CMed did not influence these genes. The proliferation of TGF-ß1-induced human cardiac fibroblasts was restored by both FGF-2 (p = 0.0002) and ASC-CMed (p = 0.0121). The present study supports the anti-fibrotic effects of FGF-2 through the blockage of cardiac fibroblast differentiation into myofibroblasts. ASC-CMed, however, did not replicate the anti-fibrotic effects of FGF-2 in vitro.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/citologia , Miofibroblastos/citologia , Fator de Crescimento Transformador beta1/farmacologia , Células Cultivadas , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Transdução de SinaisRESUMO
SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor ß1 (TGF-ß1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-ß1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-ß receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-ß1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-ß1 treated embryos. These results suggest an early action of exogenous TGF-ß1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-ß signalling during early embryogenesis.