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1.
FASEB J ; 35(9): e21845, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369625

RESUMO

Serine protease inhibitor-E2 (SERPINE2) is highly expressed in the granulosa cells of growing follicles and the dynamic changes in SERPINE2 expression are correlated with follicular development and ovulation in several mammals, including mice, cattle, sheep, and humans. Bone morphogenetic proteins (BMPs) and their functional receptors are extensively expressed in the ovary and play critical roles in the regulation of ovarian folliculogenesis and luteal function. To date, whether BMPs regulate the expression of SERPINE2 during human follicular development remains to be elucidated. The aim of this study was to investigate the effects of BMPs on the regulation of SERPINE2 expression (a major regulator of plasminogen activators [PA]) and the underlying mechanisms using primary and immortalized human granulosa-lutein (hGL) cells. Our results demonstrated that these BMPs (BMP2, BMP4, BMP6, BMP7, and BMP15) induced differential upregulation of SERPINE2 expression. In this regard, BMP2 is the major modulator that has the best cellular activity, which further decreased the production of urokinase PA and tissue PA in hGL cells. In addition to canonical SMAD1/5/8 signaling, BMP2 also activates noncanonical SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) signaling. Using two inhibition approaches (kinase receptor inhibitors and siRNA-mediated knockdown), we found that SMAD2/3-SMAD4 and p38 MAPK, but not SMAD1/5/8 signaling, was involved in the BMP2-induced upregulation of SERPINE2 expression via activin receptor-like kinase 3. These findings deepen our understanding of the differential effect of BMPs in regulating follicular function and provide new insights of the molecular mechanisms by which BMP2 regulates the expression of SERPINE2 in human granulosa cells.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Células da Granulosa/metabolismo , Células Lúteas/metabolismo , Serpina E2/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Regulação para Cima/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Cultivadas , Feminino , Humanos , Transdução de Sinais/fisiologia
2.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360888

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.


Assuntos
Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Transdução de Sinais/genética , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad3/química , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Hipertrofia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Domínios Proteicos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Membrana Sinovial/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
3.
Mol Med Rep ; 24(4)2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34328199

RESUMO

Myocardial fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix in myocardial interstitial spaces. Myocardial fibrosis is a fundamental process in ventricular remodeling and a primary contributor to the progression of heart failure. Liquiritigenin (LQ) is a flavanone compound with anti­oxidative, anti­carcinogenic, anti­inflammatory and estrogenic properties. The present study aimed to investigate the regulatory potential of LQ treatment in a mouse model of isoprenaline (ISO)­induced cardiac fibrosis and in cultured H9C2 cardiomyocytes stimulated with angiotensin II (Ang II). The treatment of ISO­induced mice with LQ significantly decreased the levels of cardiac injury­related proteins in the serum and ECM accumulation in mouse heart tissues. LQ treatment also effectively alleviated cardiac dysfunction in ISO­treated mice. Further analyses revealed that LQ inhibited ISO­induced collagen formation and activation of the transforming growth factor­ß1 (TGF­ß1)/Smad2 and protein kinase B (AKT)/extracellular signal­regulated kinase (ERK) signaling pathways. As a major pathological event in myocardial fibrosis, the apoptosis of cardiomyocytes has been considered a key mechanism contributing to impaired left ventricle performance. The pretreatment of rat cardiomyocytes with LQ significantly reduced the apoptosis of H9C2 cells, and inhibited Ang II­induced activation of the TGF­ß1/Smad2 and AKT/ERK pathways. In conclusion, the present study revealed that LQ ameliorated ISO­induced myocardial fibrosis in mice and inhibited the apoptosis of cardiomyocytes in vitro by inhibiting the TGF­ß1/Smad2 and AKT/ERK signaling pathways. These results suggested the anti­fibrotic and cardioprotective potential of LQ in fibrosis, thus supporting the use of LQ for the management of cardiomyocyte injury and myocardial fibrosis in patients with cardiac diseases.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose/tratamento farmacológico , Flavanonas/farmacologia , Cardiopatias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Angiotensina II/toxicidade , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibrose/induzido quimicamente , Flavanonas/uso terapêutico , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Testes de Função Cardíaca/efeitos dos fármacos , Isoproterenol/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/antagonistas & inibidores , Fator de Crescimento Transformador beta1/antagonistas & inibidores
4.
Cell Prolif ; 54(7): e13074, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34101281

RESUMO

OBJECTIVES: Pulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate-derived exosomes (SA-Exo) in the angiogenesis of pulp regeneration. MATERIALS AND METHODS: We extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro-angiogenetic effects of SA-Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated. RESULTS: We firstly found that SA-Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA-Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR-26a, which is enriched in SA-Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF-ß/SMAD2/3 signalling. CONCLUSIONS: In summary, these data reveal that SA-Exo shuttled miR-26a promotes angiogenesis via TGF-ß/SMAD2/3 signalling contributing to SHED aggregate-based pulp tissue regeneration. These novel insights into SA-Exo may facilitate the development of new strategies for pulp regeneration.


Assuntos
Polpa Dentária/fisiologia , Exossomos/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Compostos de Anilina/farmacologia , Antagomirs/metabolismo , Compostos de Benzilideno/farmacologia , Diferenciação Celular/efeitos dos fármacos , Exossomos/transplante , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neovascularização Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Dente Decíduo/citologia , Fator de Crescimento Transformador beta/metabolismo
5.
Gene ; 790: 145695, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33964379

RESUMO

Hypoxia promotes the secretion of basic fibroblast growth factor (bFGF) in retinal pigment epithelium (RPE), which plays an important part in retinopathy of prematurity (ROP). This study preliminarily explored the effect of hypoxia-induced RPE-derived bFGF on the biological functions of human umbilical vein endothelial cells (HUVECs). After cell culture in hypoxia conditions, the cell viability, apoptosis, and the expressions of bFGF and vascular endothelial growth factor A (VEGFA) in human RPEs were detected by 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, western blot, RT-qPCR, or ELISA. The HUVECs were transfected with siRNA for bFGF (sibFGF) or transforming growth factor-ß1 (TGF-ß1) (siTGF-ß1) and grown in the supernatant RPE under normoxia conditions or hypoxia conditions to further determine the cell viability, migration, angiogenesis, and the expressions of TGF-ß1, p-smad2/3, and smad2/3 in the cells by performing MTT, transwell, tube formation, Western blot, or RT-qPCR. Hypoxia culture decreased the cell viability and promoted the apoptosis as well as the expressions of bFGF and VEGFA in RPEs. In both normoxia and hypoxia conditions, RPE-derived bFGF increased the cell viability, migration, angiogenesis, and the expressions of TGF-ß1 and p-smad2/3 in the HUVECs, with hypoxia-induced RPE-derived bFGF showing a stronger effect than bFGF induced by normoxia. However, sibFGF reversed the effects caused by RPE-derived bFGF. Moreover, siTGF-ß1 decreased the high cell viability, migration and angiogenesis of HUVECs, and downregulated the expressions of TGF-ß1 and phosphorylated (p)-smad2/3 upregulated by hypoxia-induced RPE-derived bFGF. Hypoxia-induced RPE-derived bFGF could promote the migration and angiogenesis of HUVECs through regulating TGF-ß1/smad2/3 pathway.


Assuntos
Movimento Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipóxia/fisiopatologia , Neovascularização Fisiológica , Epitélio Pigmentado da Retina/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Apoptose , Sobrevivência Celular , Fator 2 de Crescimento de Fibroblastos/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Epitélio Pigmentado da Retina/irrigação sanguínea , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genética
6.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947038

RESUMO

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.


Assuntos
Benzamidas/farmacologia , Células Epiteliais/efeitos dos fármacos , Fibrose Peritoneal/patologia , Peritônio/citologia , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Clorexidina/análogos & derivados , Clorexidina/toxicidade , Colágeno/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Peritoneal/induzido quimicamente , Peritônio/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores
7.
J Toxicol Sci ; 46(5): 249-253, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33952801

RESUMO

Modulation of the blood coagulation fibrinolytic system is an essential function of vascular endothelial cells. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) are major fibrinolytic regulatory proteins synthesized by vascular endothelial cells; fibrinolytic activity is dependent on the balance between these proteins. Previously, we have reported that cadmium, an initiator of ischemic heart disease, induces PAI-1 expression and suppresses fibrinolytic activity in cultured human vascular endothelial cells. However, the key molecules involved in cadmium-induced PAI-1 induction remain unclear. Herein, we investigated the contribution of Smad2 and Smad3, transcriptional factors involved in PAI-1 induction via transforming growth factor-ß, using the human vascular endothelial cell line EA.hy926 cells in culture. Our findings indicated that cadmium induces PAI-1 expression without affecting t-PA expression up to 20 µM, a non-cytotoxic concentration, and PAI-1 induction by cadmium is partly mediated via Smad2 and Smad3. This study provides a possible mechanism underlying cadmium-induced vascular disorders.


Assuntos
Cádmio/toxicidade , Células Endoteliais/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Transdução de Sinais/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/metabolismo , Veias Umbilicais/citologia
8.
Biochem Biophys Res Commun ; 559: 168-175, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33945994

RESUMO

Transforming growth factor (TGF)ß/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFß/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the -2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (-2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Gástrula/embriologia , Gástrula/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Smad2/genética , Proteína Smad3/genética , Ativação Transcricional , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
9.
Cell Biochem Funct ; 39(6): 763-770, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34028068

RESUMO

Colorectal cancer (CRC) is one of the most common malignant tumours in the world. Recent reports have revealed natural products displayed inhibition on colon cancer potential by suppressing transforming growth factor-ß/Smads induced epidermal-mesenchymal transition (EMT). In this article, 12 kinds of natural berberine analogues were screened for their effects on the inhibition of the colon cancer cells, the results showed that demethyleneberberine (DM-BBR) exhibited an interesting and potential effect on inducing the apoptosis of HCT-116 cells with drug concentrations of 6, 12 and 18 µM. Particularly, DM-BBR reversed the EMT process by inhibiting the expression of p-Smad2 and p-Smad3 in the transforming growth factor-ß/Smads signal pathway, up-regulated pro-apoptotic protein cleaved caspase-9, and blocked cell cycle at the S phase and increasing the expression of cyclin proteins P27 and P21. Taken together, these findings suggested that DM-BBR could promote apoptosis and suppress TGF-ß/Smads induced EMT in the colon cancer cells HCT-116.


Assuntos
Antineoplásicos/farmacologia , Berberina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteína Smad2/antagonistas & inibidores , Proteína Smad3/antagonistas & inibidores , Fator de Crescimento Transformador beta/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Estrutura Molecular , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas
10.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805564

RESUMO

The overactivation of Wnt/ß-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of ß-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.


Assuntos
Neoplasias do Colo/patologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos Nus , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Toxicol Appl Pharmacol ; 420: 115535, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33848516

RESUMO

Epithelial-mesenchymal transition (EMT), the epithelial cells transdifferentiation into the mesenchymal cells, has been involved in cancer metastasis. Nannocystin ax (NAN) is a cyclodepsipeptide initially isolated from Myxobacterial genus, Nannocystis sp. with anticancer activities. This study was designed to explore the effect of NAN on TGF-ß1-induced EMT in lung cancer cells. The morphological alteration was observed with a microscope. Western blotting and immunofluorescence assays were used to detect the protein expression and the localization. The adhesion and migration were evaluated by adhesion assay and wound healing assay. The mRNA expression of TGF-ß receptor type I (TßRI) was determined by real-time PCR. NAN significantly restrained TGF-ß1-induced EMT morphological changes, the protein expression of E-cadherin, N-cadherin, and Vimentin, etc. TGF-ß1 activated phosphorylation and nuclear translocation of Smad2/3 were inhibited by NAN. Furthermore, NAN suppressed adhesion and migration triggered by TGF-ß1. In addition, NAN significantly down-regulated TßRI on the transcriptional level directly. In summary, these results showed that NAN restrained TGF-ß1-induced epithelial-mesenchymal transition, migration, and adhesion in human lung cancer cells. The underlying mechanism involved the inhibition of Smad2/3 and the TßRI signaling pathway. This study reveals the new anticancer effect and mechanism of NAN.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Compostos Macrocíclicos/farmacologia , Myxococcales/química , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Células A549 , Antineoplásicos/isolamento & purificação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Depsipeptídeos/isolamento & purificação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Compostos Macrocíclicos/isolamento & purificação , Fator 1 de Elongação de Peptídeos/metabolismo , Fosforilação , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
12.
Toxins (Basel) ; 13(3)2021 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800744

RESUMO

Ochratoxin A (OTA) is a mycotoxin occurring in foods consumed by humans. Recently, there has been growing global concern regarding OTA toxicity. The main target organ of OTA is the kidney, but the mechanism underlying renal toxicity is not well known. In this study, human-derived proximal tubular epithelial cells, HK-2 cells, were used for RNA-sequencing (RNA-seq) and transcriptome analysis. In total, 3193 differentially expressed genes were identified upon treatment with 200 nM OTA in HK-2 cells; of these, 2224 were upregulated and 969 were downregulated. Transcriptome analysis revealed that OTA significantly affects hypoxia, epithelial-mesenchymal transition (EMT), apoptosis, and xenobiotic metabolism pathways in kidney cells. Quantitative real-time PCR analysis showed gene expression patterns similar to RNA-seq analysis. Expression of EMT markers (E-cadherin and fibronectin), apoptosis markers (caspase-3 and Bax), and kidney injury molecule-1 (KIM-1) was suppressed by inhibiting AhR expression using siRNA, and the related transcription factors, Smad2/3, and HIF-1α were downregulated. Smad2/3 suppression with siRNA could inhibit fibronetcin, caspase-3, Bax, and KIM-1 expression. Fibronetcin, caspase-3, Bax, and KIM-1 expression could be increased with HIF-1α suppression with siRNA. Taken together, these findings suggest that OTA-mediated kidney toxicity via the AhR-Smad2/3-HIF-1α signaling pathways leads to induction of EMT, apoptosis, and kidney injury.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Túbulos Renais Proximais/efeitos dos fármacos , Ocratoxinas/toxicidade , Receptores de Hidrocarboneto Arílico/genética , Proteína Smad2/genética , Proteína Smad3/genética , Transcriptoma , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , RNA-Seq , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
13.
Biomed Pharmacother ; 139: 111500, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33901873

RESUMO

Idiopathic pulmonary fibrosis (IPF) is the most common fatal interstitial lung disease, with limited therapeutic options. The abnormal and uncontrolled differentiation and proliferation of fibroblasts have been confirmed to play a crucial role in driving the pathogenesis of IPF. Therefore, effective and well-tolerated antifibrotic agents that interfere with fibroblasts would be an ideal treatment, but no such treatments are available. Remarkably, we found that dopamine (DA) receptor D1 (D1R) and DA receptor D2 (D2R) were both upregulated in myofibroblasts in lungs of IPF patients and a bleomycin (BLM)-induced mouse model. Then, we explored the safety and efficacy of DA, fenoldopam (FNP, a selective D1R agonist) and sumanirole (SMR, a selective D2R agonist) in reversing BLM-induced pulmonary fibrosis. Further data showed that DA receptor agonists exerted potent antifibrotic effects in BLM-induced pulmonary fibrosis by attenuating the differentiation and proliferation of fibroblasts. Detailed pathway analysis revealed that DA receptor agonists decreased the phosphorylation of Smad2 induced by TGF-ß1 in primary human lung fibroblasts (PHLFs) and IMR-90 cells. Overall, DA receptor agonists protected mice from BLM-induced pulmonary fibrosis and may be therapeutically beneficial for IPF patients in a clinical setting.


Assuntos
Antibióticos Antineoplásicos , Bleomicina , Agonistas de Dopamina/uso terapêutico , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Animais , Benzimidazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Fenoldopam/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D2/efeitos dos fármacos , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacos
14.
Mol Med Rep ; 23(6)2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33899116

RESUMO

In the process of nasal tissue remodeling, nasal fibroblasts serve an important role via myofibroblast differentiation and the production of extracellular matrix (ECM). Nasal fibroblast abnormalities can lead to conditions such as chronic rhinosinusitis. Salvianolic acid B (Sal B), a water-soluble active pharmaceutical compound extract from the root of the traditional Chinese medicine Salvia miltiorrhiza, displays antioxidative, antiproliferative and antifibrosis properties. The present study aimed to investigate the mechanism underlying the effects of Sal B on nasal polyp fibroblast (NPF) myofibroblast differentiation and ECM accumulation. Primary NPFs were obtained from nasal polyps of patients with chronic sinusitis. The proliferative and cytotoxic effects of Sal B on NPFs were evaluated by performing the Cell Counting Kit-8 assay. The Transwell assay was conducted to assess cell migration. α-smooth muscle actin (α-SMA), TGF-ß1 receptor (TßR)-I, TßR-II, Smad2/3 mRNA and protein expression levels and (p)-Smad2/3 phosphorylation levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. Type III collagen and fibronectin levels were analyzed by ELISA. The results indicated that Sal B significantly downregulated TGF-ß1-induced α-SMA, fibronectin and collagen III expression levels in NPFs. Similarly, Sal B significantly decreased TGF-ß1-induced TßR-I, TßR-II, p-Smad2/3, MMP-2 and MMP-9 mRNA and protein expression levels in NPFs. Furthermore, Sal B significantly decreased TGF-ß1-induced NPF migration. Therefore, the present study indicated that Sal B inhibited myofibroblast differentiation and ECM accumulation in nasal fibroblasts, suggesting that Sal B may inhibit nasal polyp formation via certain mechanisms.


Assuntos
Benzofuranos/farmacologia , Diferenciação Celular , Matriz Extracelular/metabolismo , Miofibroblastos/efeitos dos fármacos , Pólipos Nasais/metabolismo , Transdução de Sinais , Actinas/metabolismo , Adulto , Proliferação de Células , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Pólipos Nasais/patologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo
15.
Biochem Biophys Res Commun ; 553: 58-64, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33756346

RESUMO

Human embryonic stem cells (hESCs) have the unique feature of unlimited self-renewal and differentiation into derivatives of all three germ layers in human body, providing a powerful in vitro model for studying cell differentiation. FGF2, BMP4 and TGF-ß signaling have been shown to play crucial roles in mesendodermal differentiation of hESCs. However, their underlying molecular mechanisms and other signaling pathways potentially involved in mesendodermal differentiation of hESCs remain to be further investigated. In this study, we uncover that VEGF signaling pathway plays a critical role in the mesendodermal induction of hESCs. Treating hESCs with Lenvatinib, a pan-inhibitor of VEGF receptors (VEGFRs), impedes their mesendodermal induction. Conversely, overexpression of VEGFA165, a major human VEGF isoform, promotes the mesendodermal differentiation. Similar to the VEGFR inhibitor, MEK inhibitor PD0325901 hinders mesendodermal induction of hESCs. In contrast, overexpression of ERK2GOF, an intrinsically active ERK2 mutant, markedly reduces the inhibitory effect of the VEGFR inhibitor. Thus, the MEK-ERK cascade plays an important role for the function of VEGF signaling pathway in the mesendodermal induction of hESCs. All together, this study identifies the critical role of VEGF signaling pathway as well as potential crosstalk of VEGF signaling pathway with other known signaling pathways in mesendodermal differentiation of hESCs.


Assuntos
Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Sistema de Sinalização das MAP Quinases , Mesoderma/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad5/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
16.
J Biol Chem ; 296: 100512, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33676893

RESUMO

Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-ß (TGF-ß) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-ß signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-like kinase (NLK) regulates TGF-ß signaling via modulation of Smad2/3 phosphorylation in the linker region. TGF-ß-mediated transcriptional and cellular responses are suppressed by NLK overexpression, whereas NLK depletion exerts opposite effects. Specifically, we discovered that NLK associates with Smad3 and phosphorylates the designated serine residues located in the linker region of Smad2 and Smad3, which inhibits phosphorylation at the C terminus, thereby decreasing the duration of TGF-ß signaling. Overall, this work demonstrates that phosphorylation on the linker region of Smad2/3 by NLK counteracts the canonical phosphorylation in response to TGF-ß signals, thus providing new insight into the mechanisms governing TGF-ß signaling transduction.


Assuntos
Proteínas Serina-Treonina Quinases/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Humanos , Fosforilação , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética
17.
Mol Cell Biochem ; 476(8): 2963-2973, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33772427

RESUMO

PURPOSE: Members of the transforming growth factor (TGF)-ß superfamily play a key role in the regulation of the malignant phenotype of glioblastoma by promoting invasiveness, angiogenesis, immunosuppression, and maintaining stem cell-like properties. Betaglycan, a TGF-ß coreceptor also known as TGF-ß receptor III (TßRIII), interacts with members of the TGF-ß superfamily and acts as membrane-associated or shed molecule. Shed, soluble TßRIII (sTßRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TßRIII may improve our understanding of TGF-ß pathway activity in glioblastoma METHODS: Protein levels of TßRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TßRIII levels were assessed investigating long-term glioma cell lines (LTCs), cultured human brain-derived microvascular endothelial cells (hCMECs), glioblastoma-derived microvascular endothelial cells, and glioma-initiating cell lines (GICs). The impact of TßRIII on TGF-ß signaling was investigated, and results were validated in a xenograft mouse glioma model RESULTS: Immunohistochemistry and ex vivo single-cell gene expression profiling of glioblastoma tissue showed that TßRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TßRIII expression in vitro. Specifically, we detected sTßRIII in glioblastoma-derived microvascular endothelial cells. STßRIII facilitated TGF-ß-induced Smad2 phosphorylation in vitro and overexpression of sTßRIII in a xenograft mouse glioma model led to increased levels of Smad2 phosphorylation, increased tumor volume, and decreased survival CONCLUSIONS: These data shed light on the potential tumor-promoting role of extracellular shed TßRIII which may be released by glioblastoma endothelium with high sTßRIII levels.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinógenos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Pessoa de Meia-Idade , Prognóstico , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Proteína Smad2/genética , Taxa de Sobrevida , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Artigo em Inglês | MEDLINE | ID: mdl-33684566

RESUMO

Hepatocellular carcinoma (HCC) is one of the fastest-growing causes of cancer-related mortalities worldwide and this trend is mimicked by the surge of non-alcoholic fatty liver disease (NAFLD). Altered hepatic lipid metabolism promotes HCC development through inflammation and activation of oncogenes. GDF11 is a member of the TGF-ß superfamily and recent data have implicated GDF11 as an anti-aging factor that can alleviate high-fat diet induced obesity, hyperglycemia, insulin resistance and NAFLD. However, its role in hepatic lipid metabolism is still not fully delineated. The aim of the present study was to characterize the role of GDF11 in hepatic and HCC cells lipid accumulation. To achieve this, we performed imaging, biochemical, lipidomic, and transcriptomic analyses in primary hepatocytes and in HCC cells treated with GDF11 to study the GDF11-activated signaling pathways. GDF11 treatment rapidly triggered ALK5-dependent SMAD2/3 nuclear translocation and elevated lipid droplets in HCC cells, but not in primary hepatocytes. In HCC cells, ALK5 inhibition hampered GDF11-mediated SMAD2/3 signaling and attenuated lipid accumulation. Using ultra-high-performance liquid chromatography/mass spectrometry, we detected increased accumulation of longer acyl-chain di/tri-acylglycerols and glycerophospholipids. Unbiased transcriptomic analysis identified TGF-ß and PI3K-AKT signaling among the top pathways/cellular processes activated in GDF11 treated HCC cells. In summary, GDF11 supplementation promotes pro-lipogenic gene expression and lipid accumulation in HCC cells. Integration of our "omics" data pointed to a GDF11-induced upregulation of de novo lipogenesis through activation of ALK5/SMAD2/3/PI3K-AKT pathways. Thus, GDF11 could contribute to metabolic reprogramming and dysregulation of lipid metabolism in HCC cells, without effects on healthy hepatocytes.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Carcinoma Hepatocelular/patologia , Fatores de Diferenciação de Crescimento/metabolismo , Metabolismo dos Lipídeos , Neoplasias Hepáticas/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Humanos , Lipogênese , Proteína Smad2/metabolismo , Regulação para Cima
19.
Biomed Pharmacother ; 138: 111421, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33752061

RESUMO

Allergic asthma is one of the inflammatory diseases, which has become a major public health problem. Qu zhi qiao (QZQ), a dry and immature fruit of Citrus paradisi cv. Changshanhuyou, has various flavonoids with pharmacological properties. However, there is a knowledge gap on the pharmacological properties of QZQ on allergic asthma. Therefore, here, we explored the efficacy and mechanism of total flavonoids from QZQ (TFCH) on allergic asthma. We extracted and purified TFCH and conducted animal experiments using an Ovalbumin (OVA)-induced mice model. Bronchoalveolar lavage fluid and Swiss-Giemsa staining were used to count different inflammatory cells in allergic asthma mice. We conducted histopathology and immunohistochemistry to evaluate the changes in the lungs of allergic asthma mice. Moreover, we used ELISA assays to analyze chemokines and inflammatory cytokines. Furthermore, western blot analyses were conducted to elucidate the mechanism of TFCH on allergic asthma. We established that TFCH has anti-inflammatory effects and inhibits airway remodeling, providing a potential therapeutic strategy for allergic asthma.


Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Asma/prevenção & controle , Citrus paradisi , Flavonoides/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteína Smad2/antagonistas & inibidores , Proteína Smad3/antagonistas & inibidores , Remodelação das Vias Aéreas/fisiologia , Animais , Asma/induzido quimicamente , Asma/metabolismo , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Frutas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ovalbumina/toxicidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
20.
Chem Pharm Bull (Tokyo) ; 69(2): 178-184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33518600

RESUMO

C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) plays a crucial part in cardiovascular diseases especially the coronary artery disease. Nonetheless, it is unrevealed that whether the CTRP12 participates in the progress of cardiac fibrosis. In this study, we investigated whether CTRP12 regulates pathological myocardial fibrosis. We isolated neonatal rat cardiac fibroblasts were cultured with recombination CTRP12 followed by stimulating with Isoproterenol (ISO, 100 µM) for 24 h. Then the adenovirus were used to achieve the CTRP12-overexpressed fibroblasts. In vivo, the C57/B6 mice were subjected to recombinant human CTRP12 (0.2 µg/g/d) for 2 weeks after injected with Isoproterenol (ISO, 10 mg/kg/d for 3 d then 5 mg/kg/d for 11 d, subcutaneously (s.c.), 2 weeks) and mice were also subjected to adenovirus with P38 overexpressing system to explore the mechanism. As a result, CTRP12 significantly inhibit the transformation of cardiac fibroblasts to myofibroblasts and the transcription of cardiac fibrosis-related proteins induced by ISO in vitro. The administration of CTRP12 can effectively reduce the cardiac fibrosis and enhance the cardiac function in mice hearts. The treatment with CTRP12 did not change the expression level of phosphorylated (p)-smad2, smad4, p-extracellular regulated protein kinases 1/2 and c-Jun N-terminal kinase 1/2, but it suppressed the activation of p38. Cardiac overexpression of p38 could abolish this kind of cardioprotective effects by CTRP12. In summary, the CTRP12 protect against the ISO induced cardiac fibrosis via suppressing the p38 signal pathway.


Assuntos
Adipocinas/farmacologia , Isoproterenol/toxicidade , Transdução de Sinais/efeitos dos fármacos , Adipocinas/genética , Adipocinas/metabolismo , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Cardiopatias/etiologia , Cardiopatias/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Proteína Smad2/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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