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1.
Sci Transl Med ; 13(605)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349032

RESUMO

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.


Assuntos
Fator de Crescimento Transformador beta2 , Fator de Crescimento Transformador beta3 , Animais , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Camundongos , Isoformas de Proteínas/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/metabolismo
2.
Jt Dis Relat Surg ; 32(2): 313-322, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34145806

RESUMO

OBJECTIVES: We aimed to investigate the radiological, biomechanical, histopathological, histomorphometric, and immunohistochemical effects of different doses of vardenafil on fracture healing. MATERIALS AND METHODS: Fifty-one rats were divided into three groups. Group V5 was given 5 mg/kg/day of vardenafil; Group V10 was given 10 mg/kg/day of vardenafil; and the control group was given the same volume of saline. Six rats from each group were sacrificed on Day 14 (early period) and the remaining rats were sacrificed on Day 42 (late period). Callus/femoral volume and bone mineral density were measured using micro-computed tomography. Five femurs from each group in the late period were examined by biomechanical tests. In addition to the histopathological and histomorphometric evaluations, immunohistochemical analyses were performed to examine the levels of inducible nitric oxide synthase (iNOS), transforming growth factor-3 (TGF-ß3), and nuclear factor kappa B (NF-κB) proteins. RESULTS: Both doses of vardenafil increased primary bone volume and maximal bone fracture strength in late period, compared to the control group (p<0.05). Histological healing scores of vardenafil groups were significantly higher in early period (p<0.001). While cartilaginous callus/total callus ratio in early period was higher, callus diameter/femoral diameter ratio in late period was lower in vardenafil groups (p<0.01). The NF-κB immunopositivity in V10 group decreased in early period, compared to control group (p<0.001). The TGF-ß3 and iNOS immunopositivity increased in both V5 and V10 groups, compared to the control group in early period, but returned to normal in late period. CONCLUSION: During the first period of fracture healing process in which vasodilation is mostly required with increasing inflammation, vardenafil has ameliorating effects on the bone union and supports fracture healing.


Assuntos
Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/administração & dosagem , Dicloridrato de Vardenafila/administração & dosagem , Animais , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Modelos Animais de Doenças , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/patologia , Fêmur/diagnóstico por imagem , Fêmur/patologia , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Fator de Crescimento Transformador beta3/metabolismo , Microtomografia por Raio-X
3.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063149

RESUMO

The female reproductive system ages before any other organ system in the body. This phenomenon can have tangible clinical implications leading to infertility, miscarriages, birth defects and systemic deterioration due to estrogen loss. "Fibroinflammation" is a hallmark of aging tissues; there is an increase in inflammatory cytokines and fibrotic tissue in the aging ovarian stroma. We systematically evaluated immunomodulatory factors in human follicular fluid, which, like the stroma, is a critical ovarian microenvironment directly influencing the oocyte. Using a cytokine antibody array, we identified a unique fibroinflammatory cytokine signature in follicular fluid across an aging series of women (27.7-44.8 years). This signature (IL-3, IL-7, IL-15, TGFß1, TGFß3 and MIP-1) increased with chronologic age, was inversely correlated to anti-Müllerian hormone (AMH) levels, and was independent of body mass index (BMI). We focused on one specific protein, TGFß3, for further validation. By investigating this cytokine in human cumulus cells and ovarian tissue, we found that the age-dependent increase in TGFß3 expression was unique to the ovarian stroma but not other ovarian sub-compartments. This study broadens our understanding of inflammaging in the female reproductive system and provides a defined fibroinflammatory aging signature in follicular fluid and molecular targets in the ovary with potential clinical utility.


Assuntos
Envelhecimento/patologia , Líquido Folicular/metabolismo , Inflamação/metabolismo , Ovário/metabolismo , Adulto , Hormônio Antimülleriano/metabolismo , Índice de Massa Corporal , Células do Cúmulo/metabolismo , Citocinas/metabolismo , Feminino , Fibrose , Humanos , Folículo Ovariano/irrigação sanguínea , Folículo Ovariano/metabolismo , Células Estromais/metabolismo , Fator de Crescimento Transformador beta3/metabolismo
4.
J Cell Mol Med ; 25(7): 3498-3510, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33682288

RESUMO

Transforming growth factor beta (TGF-ß) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-ß isoforms, including TGF-ß1, TGF-ß2 and TGF-ß3, remain unclear. Here, we demonstrated that all of the three TGF-ß isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-ß isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-ß isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-ß/SMAD signalling pathway-dependent and TGF-ß/SMAD signalling pathway-independent manners. TGF-ß isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-ß1 and TGF-ß2, not TGF-ß3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-ß isoforms in the HCV-related liver disease progression.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepacivirus/crescimento & desenvolvimento , Hepatite C/virologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Sequência de Aminoácidos , Antivirais/farmacologia , Linhagem Celular Tumoral , Hepatite C/patologia , Humanos , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , RNA Viral , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta3/farmacologia , Internalização do Vírus/efeitos dos fármacos
5.
Injury ; 52(6): 1294-1299, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33472741

RESUMO

INTRODUCTION & AIMS: Non Steroidal Anti-Inflammatory drugs (NSAIDs) are potent inhibitors of post-traumatic pain. Several studies have highlighted that NSAIDs could exert a negative effect on bone healing process possibly by down-regulating chondrogenesis and endochondral ossification. The aim of the study is to explore the potential mechanism though which NSAIDs can affect chondrogenesis. M&M: Trabecular bone from the fracture site was isolated from 10 patients suffering from long bone fractures. Mesenchymal Stem Cells (MSCs) were isolated following collagenase digestion and functional assays to assess the effect of diclofenac sodium on chondrogenesis were performed. Gene expression analysis of 84 key molecules was performed. RESULTS: Diclofenac sodium inhibits chondrogenic differentiation and induces a strong inhibition of prostaglandin E-2 (PGE-2) production during chondrogenic differentiation. Replenishment of PGE-2 did not reverse this negative effect. Chondrogenic inhibition is similar in cells treated only for the first week of chondrogenic differentiation or continuously for 3 weeks. Gene analysis shows a strong downregulation of TGF-ß3 and FGF-1 while TNF was upregulated. CONCLUSION: NSAIDs seem to affect the transition phase of mesenchymal stem cells towards functional chondrocytes. This effect is unrelated to the endogenous production of PGE-2. The downregulation of the expression of key molecules like TGF-ß3 seem to be the underlying mechanism.


Assuntos
Osteogênese , Fator de Crescimento Transformador beta3 , Anti-Inflamatórios não Esteroides/farmacologia , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Regulação para Baixo , Humanos , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta3/farmacologia
6.
Int J Biol Macromol ; 172: 381-393, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33476613

RESUMO

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-ß3) encapsulated (NPTGF-ß3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-ß3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.


Assuntos
Resinas Acrílicas/química , Alginatos/química , Materiais Biocompatíveis/química , Cartilagem Articular/efeitos dos fármacos , Hidrogéis/química , Nanopartículas/química , Fator de Crescimento Transformador beta3/farmacocinética , Implantes Absorvíveis , Animais , Materiais Biocompatíveis/farmacologia , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/lesões , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Composição de Medicamentos/métodos , Membro Posterior/efeitos dos fármacos , Masculino , Nanopartículas/ultraestrutura , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta3/química , Fator de Crescimento Transformador beta3/metabolismo , Resultado do Tratamento
7.
Exp Hematol ; 93: 70-84.e4, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33166613

RESUMO

Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor ß (TGFß) pathway, regulated by the TGFß1, TGFß2, and TGFß3 ligands. Accordingly, the TGFß pathway is an attractive therapeutic target for FA. While inhibition of TGFß1 and TGFß3 promotes blood cell expansion, inhibition of TGFß2 is known to suppress hematopoiesis. Here, we report the effects of AVID200, a potent TGFß1- and TGFß3-specific inhibitor, on FA hematopoiesis. AVID200 promoted the survival of murine FA HSPCs in vitro. AVID200 also promoted in vitro the survival of human HSPCs from patients with FA, with the strongest effect in patients progressing to severe aplastic anemia or myelodysplastic syndrome (MDS). Previous studies have indicated that the toxic upregulation of the nonhomologous end-joining (NHEJ) pathway accounts, at least in part, for the poor growth of FA HSPCs. AVID200 downregulated the expression of NHEJ-related genes and reduced DNA damage in primary FA HSPC in vitro and in in vivo models. Collectively, AVID200 exhibits activity in FA mouse and human preclinical models. AVID200 may therefore provide a therapeutic approach to improving BMF in FA.


Assuntos
Anemia de Fanconi/tratamento farmacológico , Hematopoese/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta3/antagonistas & inibidores , Adolescente , Adulto , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Anemia de Fanconi/metabolismo , Anemia de Fanconi/fisiopatologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo
8.
Aging (Albany NY) ; 12(24): 25564-25580, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33264103

RESUMO

The Wnt/ß-catenin pathway plays vital roles in diverse biological processes, including cell differentiation, proliferation, migration, and insulin sensitivity. A recent study reported that the DNA-binding transcriptional factor SIX3 is essential during embryonic development in vertebrates and capable of downregulating target genes of the Wnt/ß-catenin pathway in lung cancer, indicating negative regulation of Wnt/ß-catenin activation. However, regulation of the SIX3-Wnt/ß-catenin pathway axis remains unknown. We measured the expression of TRIM27 and SIX3 as well as investigated whether there was a correlation between them in lung cancer tissue samples. Herein, we found that the E3 ubiquitin ligase, TRIM27, ubiquitinates, and degrades SIX3. TRIM27 induces non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the expression of ß-catenin, S100P, TGFB3, and MMP-9 were significantly inhibited by SIX3. Furthermore, XAV939 is a selective ß-catenin-mediated transcription inhibitor that inhibited TRIM27- and SIX3-mediated NSCLC cell proliferation, migration, and invasion. Clinically, lung tissue samples of cancer patients showed increased TRIM27 expression and decreased SIX3 expression. Taken together, these data demonstrate that TRIM27 acts as an oncogene regulating cell proliferation and metastasis in NSCLC through SIX3-ß-catenin signaling.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Células A549 , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Oncogenes , Transdução de Sinais , Fator de Crescimento Transformador beta3/efeitos dos fármacos , Fator de Crescimento Transformador beta3/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , beta Catenina/efeitos dos fármacos , beta Catenina/metabolismo
9.
J Cell Mol Med ; 24(19): 11546-11557, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32845082

RESUMO

We aimed to investigate the role of the miR-29b and its effect on TGF-ß3 pathway in vascular and valvular calcification in a rat model of calcific aortic valve diseases (CAVD). A rat model of CAVD was established by administration of warfarin plus vitamin K. The expression levels of miR-29b, osteogenic markers and other genes were determined by qRT-PCR, Western blot and/or immunofluorescence and immunohistochemistry. The calcium content and alkaline phosphatase (ALP) activity were measured. The calcium content, ALP activity and osteogenic markers levels in calcified aorta and aortic valve were augmented compared to controls. The expression of miR-29b, p-Smad3, and Wnt3 and ß-catenin was significantly up-regulated, whereas TGF-ß3 was markedly down-regulated. However, compared with the CAVD model group, the calcium content and ALP activity in rats treated with antagomiR-29b were significantly decreased, and antagomiR-29b administration reversed the effects of CAVD model on the expression of miR-29b and osteogenic markers. Inhibition of miR-29b in CAVD rats prevented from vascular and valvular calcification and induced TGF-ß3 expression, suggesting that the miR-29b/TGF-ß3 axis may play a regulatory role in the pathogenesis of vascular and valvular calcification and could play a significant role in the treatment of CAVD and other cardiovascular diseases.


Assuntos
Antagomirs/uso terapêutico , Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/fisiopatologia , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Calcinose/fisiopatologia , Coração/fisiopatologia , MicroRNAs/antagonistas & inibidores , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/fisiopatologia , Animais , Antagomirs/farmacologia , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/genética , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Calcinose/genética , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/metabolismo , Ratos Sprague-Dawley , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Varfarina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
10.
Biochem Biophys Res Commun ; 530(4): 725-731, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32782154

RESUMO

Clinically deficient cartilage is difficult to regenerate, and the availability of chondrocytes is very limited. However, human adipose-derived stem cells (ADSCs) can be obtained easily and in sufficient quantities. Therefore, we will find a way of replacing chondrocytes with fat stem cells to solve the problem of seed cell origin. Previous studies have revealed that transforming growth factor-ß (TGF-ß) can promote chondrocyte differentiation and maturation. In this study, we found that TGF-ß3 in the transforming growth factor family can effectively promote the transformation process from fat stem cells to chondrocytes, thus promoting chondrogenesis. At the same time, we also further reviewed and considered the mechanism of this process. Through flow cytometry, immunohistochemical, fluorescent microscopy, qRCR, Wb etc., we found that TGF-ß3 mainly plays a role through wnt5a/ß-catenin, promoting human fat stem cell growing into the cartilage. This discovery is expected to provide new ideas in the field of cartilage regeneration.


Assuntos
Condrócitos/citologia , Condrogênese , Células-Tronco Mesenquimais/citologia , Fator de Crescimento Transformador beta3/metabolismo , Agrecanas/análise , Agrecanas/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual
11.
BMC Biotechnol ; 20(1): 48, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32854680

RESUMO

BACKGROUND: Human TGF-ß3 has been used in many studies to induce genes coding for typical cartilage matrix components and accelerate chondrogenic differentiation, making it the standard constituent in most cultivation media used for the assessment of chondrogenesis associated with various stem cell types on carrier matrices. However, in vivo data suggests that TGF-ß3 and its other isoforms also induce endochondral and intramembranous osteogenesis in non-primate species to other mammals. Based on previously demonstrated improved articular cartilage induction by a using hTGF-ß3 and hBMP-6 together on hADSC cultures and the interaction of TGF- ß with matrix in vivo, the present study investigates the interaction of a chitosan scaffold as polyanionic polysaccharide with both growth factors. The study analyzes the difference between chondrogenic differentiation that leads to stable hyaline cartilage and the endochondral ossification route that ends in hypertrophy by extending the usual panel of investigated gene expression and stringent employment of quantitative PCR. RESULTS: By assessing the viability, proliferation, matrix formation and gene expression patterns it is shown that hTGF-ß3 + hBMP-6 promotes improved hyaline articular cartilage formation in a chitosan scaffold in which ACAN with Col2A1 and not Col1A1 nor Col10A1 where highly expressed both at a transcriptional and translational level. Inversely, hTGF-ß3 alone tended towards endochondral bone formation showing according protein and gene expression patterns. CONCLUSION: These findings demonstrate that clinical therapies should consider using hTGF-ß3 + hBMP-6 in articular cartilage regeneration therapies as the synergistic interaction of these morphogens seems to ensure and maintain proper hyaline articular cartilage matrix formation counteracting degeneration to fibrous tissue or ossification. These effects are produced by interaction of the growth factors with the polysaccharide matrix.


Assuntos
Proteína Morfogenética Óssea 6/metabolismo , Cartilagem Articular/metabolismo , Quitosana/metabolismo , Medicina Regenerativa/métodos , Fator de Crescimento Transformador beta3/metabolismo , Animais , Proteína Morfogenética Óssea 6/genética , Cartilagem Articular/citologia , Diferenciação Celular , Proliferação de Células , Condrogênese/fisiologia , Colágeno , Colágeno Tipo X , Expressão Gênica , Humanos , Células-Tronco Mesenquimais , Osteogênese , Células-Tronco , Tecidos Suporte , Fator de Crescimento Transformador beta3/genética
12.
Biochim Biophys Acta Proteins Proteom ; 1868(11): 140485, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32652126

RESUMO

The proper orchestration of transforming growth factor beta (TGFß) mediated signal transduction depends upon a delicate set of interactions between specific ligands and their receptors. Here we present an in-depth profiling of the binding mechanism of TGFß3 ligand with its type II and type I receptors (TßRII and TßRI) using isothermal titration calorimetry (ITC). Studies were carried out in acidic pH as it has great physiological relevance for TGFß3 activity. Our findings reveal an unusual positive enthalpy (∆H) compensated by a large favourable entropy (∆S) during TGFß3-TßRII interaction. In addition to the hydrophobic effect, we propose that a distinct conformational switch from "closed" to "open" form as experienced by TGFß3 on binding to TßRII is contributing significantly to the increase in overall entropy of the system. Binding studies of TGFß3 and TßRII were carried out at different pH values and salt concentrations to gain further insight into the thermodynamics of the interaction. Furthermore, the importance of hydrophobic interactions on the binding affinity of TßRII with TGFß3 was confirmed by two TßRII variants (interfacial). Finally, a distinct shift from entropy to enthalpy dominated interaction was observed upon recruitment of TßRI to the binary complex forming the ternary complex.


Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Dicroísmo Circular , Escherichia coli/genética , Ligantes , Ligação Proteica , Transdução de Sinais , Espectrometria de Fluorescência , Termodinâmica , Fator de Crescimento Transformador beta3/genética
13.
J Dermatol Sci ; 99(2): 100-108, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32620316

RESUMO

BACKGROUND: Ultraviolet radiation (UVR) is the most well-known cause of skin pigmentation accompanied with photoaging. Transforming growth factor (TGF)-ß1 was previously shown to have anti-melanogenic property; however, it can induce scarring in skin. OBJECTIVE: We investigated the effect of TGF-ß3 on melanogenesis in human melanocytes cocultured with UV-irradiated skin constituent cells, and UV-irradiated human skin. METHODS: UVB irradiation or treatment with stem cell factor (SCF) and endothelin-1 (ET-1) was applied to human melanocytes cocultured with keratinocytes and/or fibroblasts and ex vivo human skin. Mechanistic pathways were further explored after treatment with TGF-ß3. RESULTS: While UVB irradiation or SCF/ET-1 enhanced melanogenesis, TGF-ß3 effectively inhibited melanin accumulation and tyrosinase activity via downregulation of the extracellular signal-regulated kinase (ERK)/microphthalmia-associated transcription factor (MITF) pathway. TGF-ß3 increased the expression of differentiation markers of keratinocytes. CONCLUSION: TGF-ß3 effectively suppressed UVR-stimulated melanogenesis indicating that topical TGF-ß3 may be a suitable candidate for the treatment of UV-associated hyperpigmentation disorders.


Assuntos
Melaninas/biossíntese , Melanócitos/metabolismo , Pele/patologia , Fator de Crescimento Transformador beta3/metabolismo , Raios Ultravioleta/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Ensaios Enzimáticos , Fibroblastos , Humanos , Queratinócitos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Melaninas/análise , Melanócitos/efeitos da radiação , Monofenol Mono-Oxigenase/metabolismo , Cultura Primária de Células , Pele/citologia , Pele/efeitos da radiação , Pigmentação da Pele/efeitos da radiação
14.
Med Sci Monit ; 26: e922029, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32447340

RESUMO

BACKGROUND Renal fibrosis occurs in the end-stage of all chronic kidney disease. Transforming growth factor-ß1 (TGF-ß1) is a central contributor in fibrosis. Identifying effective biomarkers that targets TGF-ß1 is necessary for the development of therapeutic agents for kidney disease. In this study, we investigated the effects and mechanism of long non-coding RNA (LncRNA)-ATB in TGF-ß1 induced human kidney 2 (HK-2) cells. MATERIAL AND METHODS We investigated the effects of either overexpression or knockdown of LncRNA-ATB on inflammation, cell apoptosis, and senescence in TGF-ß1 induced HK-2 cells. TGF-ß1 induced HK-2 cells served as the cell model. The gene level was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and protein expressions by western blot. Cell Counting Kit-8 (CCK-8) assay was performed for assessment of cell viability. Flow cytometry was applied for detection of cell apoptosis. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1ß, and IL-6 were measured by corresponding kits. RESULTS LncRNA-ATB was highly expressed in TGF-ß1 induced HK-2 cells. Inflammation, cell apoptosis, and senescence were enhanced by TGF-ß1 and these effects were all reduced by knockdown of LncRNA-ATB. Whereas overexpression of LncRNA-ATB had the opposite effects with knockdown of LncRNA-ATB. The TGFß/SMAD2/3 signaling pathway was activated by TGF-ß1 and this effect was further enhanced by LncRNA-ATB overexpression. Silencing LncRNA-ATB inhibited the TGFß/SMAD2/3 signaling pathway in TGF-ß1 induced cells. The effects of LncRNA-ATB overexpression aforementioned in TGF-ß1 induced cells were abolished by blockage of the TGFß/S0MAD2/3 signaling pathway. CONCLUSIONS LncRNA-ATB overexpression have promoting effects on inflammation, cell apoptosis and senescence in TGF-ß1 induced HK-2 cells via activating the TGFß/SMAD2/3 signaling pathway. LncRNA-ATB act as a key downstream mediator via activating the TGFß/SMAD2/3 signaling pathway and silencing LncRNA-ATB might be a new strategy for chronic kidney disease treatment.


Assuntos
RNA Longo não Codificante/genética , Insuficiência Renal Crônica/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Apoptose/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta3/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
Exp Eye Res ; 196: 108060, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32387619

RESUMO

Leucine-rich α-2-glycoprotein-1 (LRG1) is involved in several pathophysiological processes, including angiogenesis, cutaneous wound repair and cancer metastasis. In this study, we investigated the potential role and mechanism of LRG1 in corneal re-epithelialisation and nerve regeneration in streptozotocin-induced diabetic mice. We found decreased levels of LRG1 in the corneal epithelium after wounding in diabetic mice compared to normal controls. Hyperglycaemia downregulated the LRG1 expression in the corneal epithelium in vivo, as well as in vitro in a cultured mouse corneal epithelial stem/progenitor cell line (TKE2 cells) exposed to high glucose (HG; 30 mM) in the culture medium. Exogenous application of LRG1 accelerated corneal re-epithelialisation and nerve regeneration in normal mice and diabetic mice. LRG1 also overcame the suppression of wound healing in TKE2 cells by HG conditions, and it activated repair-related signalling by JAK2/STAT3, AKT, epidermal growth factor receptor (EGFR) and transforming growth factor (TGF)-ß3. We also found that LRG1 treatment overcame the hyperglycaemia-suppressed expression of matrix metalloproteinase 3 (MMP3) and metalloproteinase 13 (MMP13) in the regenerated corneal epithelium. The promoted effects of LRG1 on corneal re-epithelialisation and nerve regeneration were blocked by inhibitors of MMP3 and MMP13. Subconjunctival injection of 0.5 µg MMP inhibitors did not cause any obvious toxic damage in corneal epithelial cells. Immunoprecipitation and proximity ligation assay experiments confirmed that endogenous LRG1 coprecipitated with MMP3 and MMP13 in TKE2 cells. These results indicate that LRG1 promoted wound repair and nerve regeneration in the diabetic corneal epithelium by regulation of MMPs. Our findings reveal a new function and mechanism for LRG1 in the cornea, and they provide new insights for a better understanding of diabetic keratopathy.


Assuntos
Doenças da Córnea/metabolismo , Epitélio Corneano/fisiologia , Glicoproteínas/fisiologia , Metaloproteinases da Matriz/metabolismo , Regeneração Nervosa/fisiologia , Nervo Trigêmeo/fisiologia , Cicatrização/fisiologia , Animais , Células Cultivadas , Córnea/inervação , Córnea/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Receptores ErbB/metabolismo , Glucose/farmacologia , Hiperglicemia/metabolismo , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Oncogênica v-akt/metabolismo , Reepitelização , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta3/metabolismo
16.
Cancer Res ; 80(13): 2914-2926, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32366476

RESUMO

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/prevenção & controle , Neoplasias da Mama/prevenção & controle , Receptores de Estrogênio/metabolismo , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Receptores de Estrogênio/genética , Transdução de Sinais , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Elife ; 92020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32396062

RESUMO

Neuronal degeneration in the zebrafish retina stimulates Müller glia (MG) to proliferate and generate multipotent progenitors for retinal repair. Controlling this proliferation is critical to successful regeneration. Previous studies reported that retinal injury stimulates pSmad3 signaling in injury-responsive MG. Contrary to these findings, we report pSmad3 expression is restricted to quiescent MG and suppressed in injury-responsive MG. Our data indicates that Tgfb3 is the ligand responsible for regulating pSmad3 expression. Remarkably, although overexpression of either Tgfb1b or Tgfb3 can stimulate pSmad3 expression in the injured retina, only Tgfb3 inhibits injury-dependent MG proliferation; suggesting the involvement of a non-canonical Tgfb signaling pathway. Furthermore, inhibition of Alk5, PP2A or Notch signaling rescues MG proliferation in Tgfb3 overexpressing zebrafish. Finally, we report that this Tgfb3 signaling pathway is active in zebrafish MG, but not those in mice, which may contribute to the different regenerative capabilities of MG from fish and mammals.


Assuntos
Regeneração Nervosa , Neuroglia/fisiologia , Proteína Fosfatase 2/metabolismo , Retina/fisiologia , Fator de Crescimento Transformador beta3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proliferação de Células , Reprogramação Celular , Feminino , Regulação da Expressão Gênica , Masculino , Neuroglia/citologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores Notch/metabolismo , Retina/citologia , Transdução de Sinais , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta3/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
18.
J Exp Clin Cancer Res ; 39(1): 65, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293494

RESUMO

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been intensively studied in recent studies with aims of finding more concrete evidence on their mechanism of involvement in tumor progression, which is currently unknown. CAFs can secrete exosomes which are loaded with proteins, lipids and RNAs, all of which affect tumor microenvironment. The present study identified microRNA-93-5p (miR-93-5p) as a novel exosomal cargo responsible for the pro-tumorigenic effects of CAFs on colorectal cancer (CRC). METHODS: CAFs and normal fibroblasts (NFs) were isolated from cancerous tissues and matched with paracancerous tissues that had been surgically resected from CRC patients. The interaction among miR-93-5p, forkhead box A1 (FOXA1) and TGFB3 was identified through ChIP and dual luciferase reporter assays. The proliferation and apoptosis of SW480 cells co-cultured with CAFs-derived exosomes under irradiation were evaluated by CCK-8, colony formation, and flow cytometric assays. Tumorigenesis of SW480 cells in nude mice was assessed under the irradiation. RESULTS: FOXA1 was found to be associated with reduced radioresistance in CRC cells and was verified as a target of miR-93-5p. CAFs-derived exosomes contained higher miR-93-5p than those from NFs, which augmented SW480 cell proliferation and rescued them from radiation-induced apoptosis. miR-93-5p was identified as a mediator of the exosomal effects of CAFs on SW480 cells, possibly through downregulating FOXA1 and upregulating TGFB3. FOXA1 could bind to the promoter of TGFB3, thereby inhibiting nuclear accumulation of TGFB3. Also, CAFs-derived exosomes containing miR-93-5p increased the tumor growth of SW480 cells in irradiated nude mice. CONCLUSION: The present study identifies miR-93-5p as a specific exosomal cargo that rescues CRC cells against radiation-induced apoptosis.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/radioterapia , Exossomos/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Idoso , Animais , Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação para Baixo , Feminino , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Tolerância a Radiação , Fator de Crescimento Transformador beta3/genética , Regulação para Cima
19.
Mater Sci Eng C Mater Biol Appl ; 110: 110705, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32204019

RESUMO

Articular cartilage has a limited ability for self-repair after injury. Implantation of scaffolds functionalized with bioactive molecules that could induce the migration and chondrogenesis of endogenous mesenchymal stem cells (MSCs) provides a convenient alternative for in-situ cartilage regeneration. In this study, we found the synergistic effects of kartogenin (KGN) and transforming growth factor ß3 (TGF-ß3) on chondrogenesis of MSCs in vitro, indicating that KGN and TGF-ß3 are a good match for cartilage regeneration. Furthermore, we confirmed that KGN promoted the chondrogenesis of MSCs through attenuating the degradation of Runx1, which physically interacted with p-Smad3 in nuclei of MSCs. Meanwhile, we designed an injectable double-crosslinked hydrogel with superior mechanical property and longer support for cartilage regeneration by modifying sodium alginate and gelatin. When loaded with KGN conjugated polyurethane nanoparticles (PN-KGN) and TGF-ß3, this hydrogel showed biological functions by the release of KGN and TGF-ß3, which promoted the MSC migration and cartilage regeneration in one system. In conclusion, the cell-free hydrogel, along with PN-KGN and TGF-ß3, provides a promising strategy for cartilage repair by attracting endogenous MSCs and inducing chondrogenesis of recruited cells in a single-step procedure.


Assuntos
Anilidas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Hidrogéis/farmacologia , Nanopartículas/química , Ácidos Ftálicos/farmacologia , Poliuretanos/química , Regeneração/efeitos dos fármacos , Fator de Crescimento Transformador beta3/metabolismo , Alginatos/química , Anilidas/química , Animais , Cartilagem Articular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Ácido Hialurônico/química , Hidrogéis/química , Injeções/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Ácidos Ftálicos/química , Coelhos
20.
Cell Mol Neurobiol ; 40(8): 1353-1365, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32130571

RESUMO

Isoflurane postconditioning alleviates cerebral ischemic-reperfusion injury (CIRI), but the underlying mechanism has not been fully clarified. We previously demonstrated that the transforming growth factor beta-1 (TGF-ß1)/Smads signaling pathway is involved in the neuroprotective effect of isoflurane postconditioning. TGF-ß3 has a highly homologous sequence relative to that of TGF-ß1. In this study, we explored the roles of the TGF-ß3/Smad3 signaling pathway and myocyte enhancer factor 2C (MEF2C) in neuroprotection induced by isoflurane postconditioning. A CIRI rat model was established by middle cerebral artery occlusion for 1.5 h, followed by 24 h of reperfusion. Isoflurane postconditioning led to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons as compared with those of CIRI rats. Moreover, isoflurane postconditioning upregulated the expressions of TGF-ß3, p-Smad3, and MEF2C. However, the neuroprotective effect was reversed by pirfenidone, a TGF-ß3/Smad3 signaling pathway inhibitor. Also, pirfenidone treatment downregulated the expression of MEF2C. These results indicate that the TGF-ß3/Smad3 signaling pathway contributes to the neuroprotection of isoflurane postconditioning after CIRI and is possibly related to MEF2C.


Assuntos
Isoflurano/farmacologia , Fatores de Transcrição MEF2/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Fator de Crescimento Transformador beta3/metabolismo , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fatores de Transcrição MEF2/farmacologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta3/farmacologia , Regulação para Cima/efeitos dos fármacos
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