Effects of transforming growth factor-'beta' 1, interferon 'gamma' and cyclosporin A on transdifferentiation of gingival fibroblasts in myofibroblasts

Orientador: Ricardo Della ColettaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Miofibroblastos são células especializadas que exercem funções importantes em processos fibróticos através da síntese elevada de macromoléculas da matriz extracelular, proteases e citocinas. O exato mecanismo do surgimento (transdiferenciação) dos miofibroblastos permanece desconhecido, porém inúmeros estudos sugerem que o fator de crescimento transformante-'beta'1 (TGF-'beta'1) exerça um papel importante neste processo via ativação do fator de crescimento de tecido conjuntivo (CTGF). Em oposição aos efeitos de TGF-'beta'1, algumas citocinas inflamatórias, incluindo interferon 'gama' (IFN'gama'), parecem inibir a transdiferenciação dos miofibroblastos. O uso da droga imunossupressora ciclosporina A (CsA) é freqüentemente associada a inúmeros efeitos colaterais, sendo o aumento gengival fibrótico o de maior interesse para a área odontológica. Linhagens celulares de fibroblastos de gengiva normal tratadas com CsA expressam níveis elevados de TGF-'gama'1 e colágeno, que são intrínsecos do fenótipo miofibroblástico. Os objetivos deste estudo foram compreender a participação de TGF-'beta'1 e IFN'gama' na transdiferenciação dos fibroblastos gengivais em miofibroblastos e entender os mecanismos de ação destes fatores neste processo. Adicionalmente, nós verificamos a presença de miofibroblastos em aumentos gengivais induzidos por CsA. Nossos resultados demonstraram, através de 4 diferentes ensaios: transcriptase reversa-reação em cadeia da polimerase (RTPCR), western blot, imunofluorescência e citometria de fluxo, que TGF-'beta'1 induz e FNg bloqueia a transdiferenciação dos fibroblastos gengivais em miofibroblastos metabolicamente ativos em uma maneira dependente da dose e tempo. Nas células onde a expressão de CTGF foi significantemente reduzida por seqüências específicas de RNA de interferência, TGF-'beta'1 não foi capaz de promover a transdiferenciação dos miofibroblastos, revelando que TGF-'beta'1 é dependente da ativação de CTGF para induzir a transdiferenciação dos fibroblastos gengivais em miofibroblastos. IFN'gama' bloqueou a transdiferenciação dos miofibroblastos por estimular a expressão de Smad 7, uma proteína que regula negativamente a atividade de TGF-'beta'1. Interessantemente, miofibroblastos não foram associados aos aumentos gengivais induzidos por CsA, como revelado pelos modelos in vivo de injeções diárias de CsA em ratos Wistar e in vitro de tratamento de fibroblastos gengivais com CsA. Embora CsA tenha induzido a expressão de TGF-'beta'1, não demonstrou nenhum efeito na modulação dos níveis de CTGF. Nossos resultados demonstram que a indução da transdiferenciação dos miofibroblastos por TGF-'beta'1 é dependente da estimulação de CTGF. Adicionalmente, este estudo sugere que IFN'gama' pode ser clinicamente efetivo no tratamento de alterações fibróticas associadas à presença de miofibroblastos, pois atenua a excessiva produção de colágeno tipo I por estas célulasAbstract: Myofibroblasts are the main cellular type involved in extracellular matrix deposition in fibrotic diseases. Relatively little is known about the underlying mechanisms that regulate myofibroblast emergence (transdifferentiation), however the regulatory cytokine transforming growth factor-beta 1 (TGF-'beta'1) has been traditionally considered an inducer of the myofibroblastic phenotype via activation of connective tissue growth factor (CTGF)-dependent pathway. Some inflammatory cytokines, particularly interferon g (IFN'gama'), show opposite effects to TGF-'beta'1, inhibiting myofibroblast transdifferentiation. Cyclosporin A (CsA) is a widely used immunosuppressant drug that causes significant side effects such as gingival overgrowth. Normal gingival fibroblast cell lines treated with CsA express high levels of TGF-'beta'1 and collagen, which are intrinsic of myofibroblasts. In the present study we examined whether TGF-'beta'1 and IFN'gama' can modulate transdifferentiation of gingival fibroblasts into myofibroblasts, and the biologic mechanisms behind those factors in this process. Additionally, we have analyzed the presence of myofibroblasts in CsA-induced gingival overgrowth. Our results demonstrated throughout a modality of experiments that included reverse trancriptasepolymerase chain reaction (RT-PCR), western blot, immunofluorescence and flow citometry analysis, that TGF-'beta'1 induces and IFN'gama' blocks fibroblast-myofibroblast transdifferentiation in a dose- and time-dependent manner. In fibroblasts with CTGF levels know-down by specific small interference RNA, TGF-'beta'1 effect on transdifferentiation was significantly reduced, revealing that CTGF plays a crucial role in mediating TGF-'beta'1 myofibroblast transdifferentiation. IFN'gama' blocked myofibroblast transdifferentiation by stimulating Smad 7 levels, a protein that negatively regulates TGF-'beta'1 signaling. Interestingly, myofibroblasts were not found in CsA-induced gingival overgrowth, as revealed by the in vivo model of daily injections of CsA in Wistar rats and by the in vitro model of gingival fibroblast cultures treated with CsA. Although CsA treatment stimulated TGF-'beta'1 expression by NG fibroblasts, it lacked to induce CTGF levels. Our results demonstrate that TGF-'beta'1 induction of transdifferentiation of gingival fibroblasts to myofibroblasts occurs via a CTGF dependent pathway. Additionally, this study suggests that IFN'gama' may be clinically effective in the treatment of fibrotic diseases associate with myofibroblastMestradoPatologiaMestre em Estomatopatologi

Fungal metalloprotease generate whey-derived peptides that may be involved in apoptosis in B16F10 melanoma cells

Abstract Proteases are enzymes that act in the hydrolysis of proteins and have several industrial applications. Moreover, proteases have gained prominence as enzymes for the generation of bioactive peptides from the hydrolysis of different protein sources. Milk is the most studied protein source to obtain peptides due to its nutritional and physiological effects and has been studied as complementary therapeutic approaches for the cancer treatment, interacting specifically with cancer cells, consequently fewer side effects. The ability of Eupenicillium javanicum metalloprotease to generate whey-derived peptides with antioxidant activity has already been demonstrated. For this reason, we thus hypothesized that whey-derived peptides from Eupenicillium javanicum metalloprotease hydrolysis could also have a potential against melanoma cell lines. In this study, B16F10 melanoma cells were treated for 72 h with whey-derived peptides and the effects on cell viability were determined. Moreover, the protein profiles of the treated and nontreated cells were compared in proteomic assay and mass spectrometry analyzes. Whey-derived peptides impaired about 62% cell viability, and proteomic approach associated this behavior to modulate proteins involved in proliferation, energy, apoptosis, metastatic and malignancy rates. This study describes the relevance of microbial enzymes in generation of whey-derived peptides with biological activity against melanoma cells

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.Peer reviewe

Smad7 and CTGF particapation on gingival myofibroblasts transdifferentation and analysis of myofibroblasts influence on oral squamous cell carcinoma proliferation and invasion

Orientador: Ricardo Della ColettaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Miofibroblastos são células mesenquimais caracterizadas pela expressão da isoforma ? da actina de músculo liso (?-SMA) e pela secreção de proteínas da matriz extracelular, fatores de crescimento e proteases. Estas células desempenham um papel importante na reparação de feridas e em processos patológicos, incluindo fibroses e cânceres. Os objetivos deste estudo foram 1) analisar o papel do fator de crescimento de tecido conjuntivo bem como o efeito da superexpressão de Smad7 na transdiferenciação de miofibroblastos gengivais induzida pelo fator de crescimento transformante-?1 (TGF-?1), 2) isolar e caracterizar linhagens celulares de miofibroblastos do estroma de carcinomas espinocelulares (CEC) orais e comparar o potencial proliferativo e produção de metaloproteinases de matriz (MMP) com linhagens celulares de fibroblastos do estroma de CEC orais, e 3) analisar a influência de miofibroblastos na modulação da proliferação e invasão de linhagens celulares de CEC oral. Nossos resultados demonstraram que o tratamento com TGF-?1 induziu simultaneamente a expressão de ?-SMA e CTGF e a neutralização de CTGF com RNA de interferência (siRNA) bloqueou o efeito de TGF-?1 na indução da transdiferenciação de células de gengiva normal em miofibroblastos. A superexpressão de Smad7 em células de GN inibiu a cascata de ativação de TGF-?1, caracterizada pela fosforilação de Smad2 e expressão de ?-SMA, CTGF e colágeno tipo I. Similarmente, miofibroblastos isolados do tecido gengival de fibromatose gengival hereditária (FGH) superexpressando Smad7 demonstraram níveis reduzidos de ?-SMA e pSmad2, além de baixos níveis de expressão de CTGF e colágeno tipo I. Três linhagens celulares de miofibroblastos foram isoladas do estroma de CEC de língua e caracterizadas pela expressão de ?-SMA e por níveis elevados de produção de colágeno tipo I. Embora o potencial proliferativo dos clones de fibroblastos e miofibroblastos tenham sido semelhantes, as produções de MMP-1, -2, -9 e -13 foram significantemente maiores em miofibroblastos. Finalmente, nós demonstramos que miofibroblastos do estroma tumoral produzem níveis elevados de alguns fatores de crescimento comparado com fibroblastos, incluindo ativina A. Meios de cultura condicionados por miofibroblastos contendo ativina A significantemente induziu a proliferação de linhagens celulares de CEC oral e uma maior progressão tumoral in vivo, enquanto que o bloqueio de ativina A por siRNA diminuiu significantemente a proliferação das células de CEC oral. In vitro, miofibroblastos induziram a invasão de linhagens celulares de CEC oral, o qual foi acompanhado por uma indução na produção de MMPs, e in vivo uma significante correlação entre presença de miofibroblastos e atividades de MMP-2 e MMP-9 foi observada. O bloqueio da síntese de ativina A por siRNA em miofibroblastos não alterou a capacidade de indução da invasão e síntese de MMPs. Os resultados deste estudo demonstram 1) que Smad7 bloqueia a transdiferenciação de miofibroblastos gengivais por meio da inibição da fosforilação de Smad2 e da transcrição de CTGF, 2) que miofibroblastos no estroma de CEC orais podem contribuir para um fenótipo mais invasivo via secreção de elevados níveis de MMPs e 3) que produtos de síntese dos miofibroblastos induzem a proliferação e invasão das células de CEC oral e os estímulos proliferativos são controlados pela produção de ativina A.Abstract: Myofibroblasts are mesenchymal cells, characterized by the specific isoform ? of the smooth muscle actin (?-SMA) expression and the extracellular matrix proteins, growth factors and proteases secretion. These cells play a central role on wound healings and in pathologic process, including fibrosis and cancers. The aims of this study were 1) analyze the connective tissue growth factor (CTGF) role and the superexpression of Smad7 effect on TGF-?1-induced gingival myofibroblasts transdifferentiation, 2) isolate and characterize myofibroblast cell lines from oral squamous cell carcinomas stroma (OSCC) and compare the proliferative potential and matrix metalloproteinases (MMP) production with fibroblast cell lines from OSCCs stroma, and 3) analyze the myofibroblasts influence on the modulation of OSCC cell lines proliferation and invasion. Our results demonstrated that the TGF-?1 treatment induced simultaneously the ?-SMA and CTGF expression and the CTGF neutralization using the small interference RNA (siRNA) blocked the TGF-?1-induced gingival myofibroblasts transdifferentiation. Smad 7 superexpression in normal gingival cells (NG) inhibit the TGF-?1 cascade activation, characterized by the Smad2 phosphorilation and ?-SMA, CTGF and type I collagen expression. Similarly, hereditary gingival fibromatosis (HGF) myofibroblasts superexpressing Smad 7, demonstrated reduced levels of ?-SMA and phospho-Smad2, and low expression levels of CTGF and type I collagen. Three myofibroblast cell lines were isolated from tongue OSCC stroma and characterized by the ?-SMA expression and high levels of type I collagen. Although the proliferative potential of fibroblast and myofibroblast clones has been similar, the MMP-1, -2, -9 and -13 were significantly higher in myofibroblasts. Finally, we demonstrated that tumor stroma myofibroblasts produce high levels of some growth factors compared with fibroblasts, including activin A. Myofibroblasts conditioned medium containing activin A induce significantly the OSCC cell lines proliferation and a tumor progression in vivo, while the activin A dowregulation by siRNA significantly decreased the OSCC cells proliferation. In vitro, myofibroblasts induced OSCC cells invasion, accompanied by an induction of MMPs production, and in vivo was observed a significant correlation between the myofibroblasts presence and the MMP-2 and MMP-9 activity. The myofibroblasts dowregulation of activin A by siRNA did not affect the induction of invasion and MMPs synthesis. The results of this study demonstrate that 1) Smad 7 blockage the gingival myofibroblasts transdifferantiation through the inhibition of Smad 2 phosphorilation and CTGF transcription, 2) myofibroblasts on the OSCCs stroma can contribute to a more invasive phenotype via elevated levels of MMPs secretion, 3) myofibroblasts released products induce an OSCC cells proliferation and invasion and the proliferative stimulus are controlled by the activin A production.DoutoradoEstomatologiaDoutor em Estomatopatologi

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation

Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-β1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-β1 expression by NG fibroblasts, it lacked to induce expression and production of isoform α of smooth muscle actin (α-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-β1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression

Downregulation of activin A leads to a decrease in proliferation.

<p>Treatment with recombinant activin A was not able to promote proliferation of HaCat cells, as revealed by bromodeoxyuridine (BrdU) incorporation index (A) and cell cycle analysis (B). (C) Follistatin at 100 ng/ml significantly blocked BrdU incorporation in SCC-9 ZsGreen LN-1 cells, but no effects on cell cycle distribution were observed (D). Knockdown of activin A significantly decreased proliferation (E), enhancing the number of cells at G0/G1 and reducing the number in the S phase of cell cycle (F). Bars represent the means ± SD of three independent experiments. *p<0.01, **p<0.001, ***p<0.0001.</p