RESUMO
Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.
Assuntos
Diferenciação Celular , Osteoblastos , Osteogênese , Ligamento Periodontal , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Diferenciação Celular/genética , Células Cultivadas , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor ß (TGFß) is present in blood of patients who do not respond to anti-programmed cell death (ligand) 1 [PD-(L)1] treatment, and through synergy with vascular endothelial growth factor (VEGF), it helps to create an environment that promotes tumor immune evasion and immune tolerance. Therefore, simultaneous inhibition of TGFß/VEGF is more effective than targeting TGFß alone. In this study, the dual inhibitory mechanism of TU2218 was identified through in vitro analysis mimicking the tumor microenvironment, and its antitumor effects were analyzed using mouse syngeneic tumor models. TU2218 directly restored the activity of damaged cytotoxic T lymphocytes (CTLs) and natural killer cells inhibited by TGFß and suppressed the activity and viability of regulatory T cells. The inactivation of endothelial cells induced by VEGF stimulation was completely ameliorated by TU2218, an effect not observed with vactosertib, which inhibits only TGFß signaling. The combination of TU2218 and anti-PD1 therapy had a significantly greater antitumor effect than either drug alone in the poorly immunogenic B16F10 syngeneic tumor model. The mechanism of tumor reduction was confirmed by flow cytometry, which showed upregulated VCAM-1 expression in vascular cells and increased influx of CD8 + CTLs into the tumor. As another strategy, combination of anti-CTLA4 therapy and TU2218 resulted in high complete regression (CR) rates in CT26 and WEHI-164 tumor models. In particular, immunological memory generated by the combination of anti-CTLA4 and TU2218 in the CT26 model prevented the development of tumors after additional tumor cell transplantation, suggesting that the TU2218-based combination has therapeutic potential in immunotherapy.
Assuntos
Inibidores de Checkpoint Imunológico , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Humanos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Feminino , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linhagem Celular Tumoral , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Imunoterapia/métodosRESUMO
Rationale: Acute kidney injury (AKI) has substantial rates of mortality and morbidity, coupled with an absence of efficacious treatment options. AKI commonly transits into chronic kidney disease (CKD) and ultimately culminates in end-stage renal failure. The interferon-stimulated gene 15 (ISG15) level was upregulated in the kidneys of mice injured by ischemia-reperfusion injury (IRI), cisplatin, or unilateral ureteral obstruction (UUO), however, its role in AKI development and subsequent AKI-to-CKD transition remains unknown. Methods: Isg15 knockout (Isg15 KO) mice challenged with bilateral or unilateral IRI, cisplatin, or UUO were used to investigate its role in AKI. We established cellular models with overexpression or knockout of ISG15 and subjected them to hypoxia-reoxygenation, cisplatin, or transforming growth factor- ß1 (TGF-ß1) stimulation. Renal RNA-seq data obtained from AKI models sourced from public databases and our studies, were utilized to examine the expression profiles of ISG15 and its associated genes. Additionally, published single cell RNA-seq data from human kidney allograft biopsies and mouse IRI model were analyzed to investigate the expression patterns of ISG15 and the type I TGF-ß receptor (TGFßR1). Western blotting, qPCR, co-immunoprecipitation, and immunohistochemical staining assays were performed to validate our findings. Results: Alleviated pathological injury and renal function were observed in Isg15 KO mice with IRI-, cisplatin-, or UUO-induced AKI and the following AKI-to-CKD transition. In hypoxia-reoxygenation, cisplatin or TGF-ß1 treated HK-2 cells, knockout ISG15 reduced stimulus-induced cell fibrosis, while overexpression of ISG15 with modification capacity exacerbated cell fibrosis. Immunoprecipitation assays demonstrated that ISG15 promoted ISGylation of TGFßR1, and inhibited its ubiquitination. Moreover, knockout of TGFßR1 blocked ISG15's fibrosis-exacerbating effect in HK-2 cells, while overexpression of TGFßR1 abolished the renal protective effect of ISG15 knockout during IRI-induced kidney injury. Conclusions: ISG15 plays an important role in the development of AKI and subsequent AKI-to-CKD transition by promoting TGFßR1 ISGylation.
Assuntos
Injúria Renal Aguda , Cisplatino , Citocinas , Camundongos Knockout , Traumatismo por Reperfusão , Ubiquitinas , Animais , Humanos , Masculino , Camundongos , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Cisplatino/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Ubiquitinas/metabolismo , Ubiquitinas/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/complicações , Obstrução Ureteral/genéticaAssuntos
Proliferação de Células , Osteossarcoma , Humanos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/imunologia , Proliferação de Células/efeitos dos fármacos , Animais , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Camundongos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Administração OralRESUMO
Nineteen flavonoids were isolated from the fruits of Psoralea corylifolia L., including a novel flavanol (3) and three novel isoflavones (12-14). Their chemical structures were unequivocally determined through comprehensive spectral data analysis. The anti-proliferative effect of the isolated flavonoids was assessed in vitro using the MTT assay. Molecular docking and ELISA were employed to determine the inhibitory effects of the active compounds on ALK5. Isobavachalcone was found to inhibit TGF-ß1 induced EMT in A549 cells by Wound healing assay and Transwell chamber assay. Immunofluorescence assay and Western blot assay showed that IBC could inhibit cytoskeleton rearrangement, reduce the phosphorylation of ALK5, ERK, and Smad, down-regulate Snail expression, and up-regulate E-cadherin expression in TGF-ß1 induced A549 cells, thereby exerting the potential inhibitory effects on epithelial-mesenchymal transition (EMT) process in A549 cells. The findings presented herein establish a fundamental basis for investigating the anti-proliferative and anti-metastatic properties of psoralen flavonoids in human non-small cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Transição Epitelial-Mesenquimal , Flavonoides , Frutas , Neoplasias Pulmonares , Simulação de Acoplamento Molecular , Psoralea , Humanos , Células A549 , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Flavonoides/farmacologia , Flavonoides/química , Flavonoides/isolamento & purificação , Frutas/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Estrutura Molecular , Psoralea/química , Receptor do Fator de Crescimento Transformador beta Tipo I , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Isoflavonas/química , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologiaRESUMO
TGF-ß1/Smads is a classic signaling pathway, which plays important roles in the development process of organisms. Black porgy Acanthopagrus schlegelii and red porgy Pagrus major are valuable economic fishes, and their hybrid offspring show excellent heterosis traits. Yet the molecular regulation mechanism of the heterosis traits is less clear. Here, we explored the TGF-ß1/Smads pathway's molecular genetic information for heterosis in A. schlegelii â × P. major â (AP) and A. schlegelii â × P. major â (PA) in terms of growth and development. The mRNA expression levels of TGF-ß1, TßR-I, TßR-II, and Smad2 genes in different developmental stages of A. schlegelii were detected. Furthermore, the expression levels of TGF-ß1, TßR-I, TßR-II, and Smad2 genes in different tissues of adult (mRNA level) and larva (mRNA and protein level) of A. schlegelii, P. major, and their hybrids were determined by both real-time quantitative PCR and Western blot techniques. The results indicated the ubiquitous expression of these genes in all developmental stages of A. schlegelii and in all tested tissues of A. schlegelii, P. major, and its hybrids. Among them, the mRNA of TGF-ß1, TßR-I, and TßR-II genes is highly expressed in the liver, gill, kidney, and muscle of black porgy, red porgy, and their hybrid offspring. There are significant changes in gene and protein expression levels in hybrid offspring, which indirectly reflect hybrid advantage. In addition, there was no correlation between protein and mRNA expression levels of Smad2 protein. The results provide novel data for the differential expression of growth and development genes between the reciprocal hybridization generation of black porgy and red porgy and its parents, which is conducive to further explaining the molecular regulation mechanism of heterosis in the growth and development of hybrid porgy.
Assuntos
Vigor Híbrido , Proteína Smad2 , Fator de Crescimento Transformador beta1 , Animais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Vigor Híbrido/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Hibridização Genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Perciformes/genética , Perciformes/crescimento & desenvolvimento , Perciformes/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Feminino , Masculino , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Neuropilin-1 (NRP1), a co-receptor for various cytokines, including TGF-ß, has been identified as a potential therapeutic target for fibrosis. However, its role and mechanism in renal fibrosis remains elusive. Here, we show that NRP1 is upregulated in distal tubular (DT) cells of patients with transplant renal insufficiency and mice with renal ischemia-reperfusion (I-R) injury. Knockout of Nrp1 reduces multiple endpoints of renal injury and fibrosis. We find that Nrp1 facilitates the binding of TNF-α to its receptor in DT cells after renal injury. This signaling results in a downregulation of lysine crotonylation of the metabolic enzyme Cox4i1, decreases cellular energetics and exacerbation of renal injury. Furthermore, by single-cell RNA-sequencing we find that Nrp1-positive DT cells secrete collagen and communicate with myofibroblasts, exacerbating acute kidney injury (AKI)-induced renal fibrosis by activating Smad3. Dual genetic deletion of Nrp1 and Tgfbr1 in DT cells better improves renal injury and fibrosis than either single knockout. Together, these results reveal that targeting of NRP1 represents a promising strategy for the treatment of AKI and subsequent chronic kidney disease.
Assuntos
Injúria Renal Aguda , Fibrose , Camundongos Knockout , Neuropilina-1 , Receptor do Fator de Crescimento Transformador beta Tipo I , Traumatismo por Reperfusão , Proteína Smad3 , Neuropilina-1/metabolismo , Neuropilina-1/genética , Animais , Humanos , Camundongos , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Injúria Renal Aguda/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Proteína Smad3/metabolismo , Proteína Smad3/genética , Masculino , Fator de Necrose Tumoral alfa/metabolismo , Transdução de Sinais , Camundongos Endogâmicos C57BL , Túbulos Renais/patologia , Túbulos Renais/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Colágeno/metabolismoRESUMO
BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a challenging lung arterial disorder with remarkably high incidence and mortality rates, and the efficiency of current HPH treatment strategies is unsatisfactory. Endothelial-to-mesenchymal transition (EndMT) in the pulmonary artery plays a crucial role in HPH. Previous studies have shown that lncRNA-H19 (H19) is involved in many cardiovascular diseases by regulating cell proliferation and differentiation but the role of H19 in EndMT in HPH has not been defined. METHODS: In this research, the expression of H19 was investigated in PAH human patients and rat models. Then, we established a hypoxia-induced HPH rat model to evaluate H19 function in HPH by Echocardiography and hemodynamic measurements. Moreover, luciferase reporter gene detection, and western blotting were used to explore the mechanism of H19. RESULTS: Here, we first found that the expression of H19 was significantly increased in the endodermis of pulmonary arteries and that H19 deficiency obviously ameliorated pulmonary vascular remodelling and right heart failure in HPH rats, and these effects were associated with inhibition of EndMT. Moreover, an analysis of luciferase activity indicated that microRNA-let-7 g (let-7 g) was a direct target of H19. H19 deficiency or let-7 g overexpression can markedly downregulate the expression of TGFßR1, a novel target gene of let-7 g. Furthermore, inhibition of TGFßR1 induced similar effects to H19 deficiency. CONCLUSIONS: In summary, our findings demonstrate that the H19/let-7 g/TGFßR1 axis is crucial in the pathogenesis of HPH by stimulating EndMT. Our study may provide new ideas for further research on HPH therapy in the near future.
Assuntos
Transição Epitelial-Mesenquimal , Hipertensão Pulmonar , MicroRNAs , RNA Endógeno Competitivo , RNA Longo não Codificante , Transdução de Sinais , Fator de Crescimento Transformador beta , Animais , Feminino , Humanos , Masculino , Ratos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/fisiologia , Transição Epitelial-Mesenquimal/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/metabolismo , Hipóxia/genética , MicroRNAs/metabolismo , MicroRNAs/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , RNA Endógeno Competitivo/genética , RNA Endógeno Competitivo/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
It has been demonstrated that an enriched environment (EE) treatment can alter neuroplasticity in neurodegenerative diseases. However, the role of EE treatment in ischemic stroke remains unclear. Previous findings have revealed that EE treatment can promote cerebral activin-receptor-like-kinase-5 (ALK5) expression after cerebral ischemia/reperfusion (I/R) injury. ALK5 has been identified as a potential mediator of neuroplasticity through its modulation of Smad2/3 and Gadd45ß. Therefore, the aim of this study was to investigate whether EE treatment could promote neurofunctional recovery by regulating the ALK5/Smad2/3/Gadd45ß pathway. The study utilized the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). The ALK5/Smad2/3/Gadd45ß signaling pathway changes were evaluated using western blotting (WB). Brain injury was assessed by infarct volume and neurobehavioral scores. The effect of EE treatment on neurogenesis was evaluated using Doublecortin (DCX) and Nestin, axonal plasticity with biotinylated dextran amine (BDA) nerve tracing, and dendritic plasticity was assessed using Golgi-Cox staining. EE treatment has been demonstrated to modulate the Smad2/3/Gadd45ß pathway by regulating the expression of ALK5. The protective effects of EE treatment on brain infarct volume, neurological function, newborn neurons, dendritic and axonal plasticity following cerebral I/R injury were counteracted by ALK5 silencing. EE treatment can enhance neurofunctional recovery after cerebral I/R injury, which is achieved by regulating the ALK5/Smad2/3/Gadd45ß signaling pathway to promote neuroplasticity.
Assuntos
Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Traumatismo por Reperfusão , Transdução de Sinais , Proteína Smad2 , Animais , Masculino , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Traumatismo por Reperfusão/metabolismo , Recuperação de Função Fisiológica/fisiologia , Proteína Duplacortina , Proteína Smad3/metabolismo , Isquemia Encefálica/metabolismo , Meio Ambiente , Infarto da Artéria Cerebral Média/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas GADD45 , Antígenos de DiferenciaçãoRESUMO
BACKGROUND: Fibrosis is a significant pathological feature of chronic skeletal muscle injury, profoundly affecting muscle regeneration. Fibro-adipogenic progenitors (FAPs) have the ability to differentiate into myofibroblasts, acting as a primary source of extracellular matrix (ECM). the process by which FAPs differentiate into myofibroblasts during chronic skeletal muscle injury remains inadequately explored. METHOD: mouse model with sciatic nerve denervated was constructed and miRNA expression profiles between the mouse model and uninjured mouse were analyzed. qRT/PCR and immunofluorescence elucidated the effect of miR-27b-3p on fibrosis in vivo and in vitro. Dual-luciferase reporter identified the target gene of miR-27b-3p, and finally knocked down or overexpressed the target gene and phosphorylation inhibition of Smad verified the influence of downstream molecules on the abundance of miR-27b-3p and fibrogenic differentiation of FAPs. RESULT: FAPs derived from a mouse model with sciatic nerves denervated exhibited a progressively worsening fibrotic phenotype over time. Introducing agomiR-27b-3p effectively suppressed fibrosis both in vitro and in vivo. MiR-27b-3p targeted Transforming Growth Factor Beta Receptor 1 (TGF-ßR1) and the abundance of miR-27b-3p was negatively regulated by TGF-ßR1/Smad. CONCLUSION: miR-27b-3p targeting the TGF-ßR1/Smad pathway is a novel mechanism for regulating fibrogenic differentiation of FAPs. Increasing abundance of miR-27b-3p, suppressing expression of TGF-ßR1 and inhibiting phosphorylation of smad3 presented potential strategies for treating fibrosis in chronic skeletal muscle injury.
Assuntos
Fibrose , MicroRNAs , Músculo Esquelético , Transdução de Sinais , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Camundongos , Doença Crônica , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Smad/metabolismo , Proteínas Smad/genética , Masculino , Modelos Animais de Doenças , Diferenciação Celular , Nervo Isquiático/lesõesRESUMO
Excessive transforming growth factor ß1 (TGF-ß1) secreted by activated hepatic stellate cells (aHSCs) aggravates liver fibrosis via over-activation of TGF-ß1-mediated signaling pathways in a TGF-ß type I receptor (TßRI) dependent manner. TßRI with the C-terminal valine truncated (RIPΔ), as a novel TßRI-mimicking peptide, is an appealing anti-fibrotic candidate by competitive binding of TGF-ß1 to block TGF-ß1 signal transduction. Platelet-derived growth factor receptor ß (PDGFßR) is highly expressed on the surface of aHSCs in liver fibrosis. Herein, we designed a novel RIPΔ variant Z-RIPΔ (PDGFßR-specific affibody ZPDGFßR fused to the N-terminus of RIPΔ) for liver fibrosis therapy, and expect to improve the anti-liver fibrosis efficacy by specifically inhibiting the TGF-ß1 activity in aHSCs. Target peptide Z-RIPΔ was prepared in Escherichia coli by SUMO fusion system. Moreover, Z-RIPΔ specifically bound to TGF-ß1-activated aHSCs, inhibited cell proliferation and migration, and reduced the expression of fibrosis markers (α-SMA and FN) and TGF-ß1 pathway-related effectors (p-Smad2/3 and p-p38) in vitro. Furthermore, Z-RIPΔ specifically targeted the fibrotic liver, alleviated the liver histopathology, mitigated the fibrosis responses, and blocked TGF-ß1-mediated Smad and p38 MAPK cascades. More importantly, Z-RIPΔ exhibited a higher fibrotic liver-targeting capacity and stronger anti-fibrotic effects than its parent RIPΔ. Besides, Z-RIPΔ showed no obvious toxicity effects in treating both an in vitro cell model and an in vivo mouse model of liver fibrosis. In conclusion, Z-RIPΔ represents a promising targeted candidate for liver fibrosis therapy.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Proteínas Smad , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por Mitógeno , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Animais , Fator de Crescimento Transformador beta1/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Smad/metabolismo , Masculino , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Humanos , Camundongos Endogâmicos C57BLRESUMO
Oral submucous fibrosis (OSF) is a chronic and inflammatory mucosal disease caused by betel quid chewing, which belongs to oral potentially malignant disorders. Abnormal fibroblast differentiation leading to disordered collagen metabolism is the core process underlying OSF development. The epithelium, which is the first line of defense against the external environment, can convert external signals into pathological signals and participate in the remodeling of the fibrotic microenvironment. However, the specific mechanisms by which the epithelium drives fibroblast differentiation remain unclear. In this study, we found that Arecoline-exposed epithelium communicated with the fibrotic microenvironment by secreting exosomes. MiR-17-5p was encapsulated in epithelial cell-derived exosomes and absorbed by fibroblasts, where it promoted cell secretion, contraction, migration and fibrogenic marker (α-SMA and collagen type I) expression. The underlying molecular mechanism involved miR-17-5p targeting Smad7 and suppressing the degradation of TGF-ß receptor 1 (TGFBR1) through the E3 ubiquitination ligase WWP1, thus facilitating downstream TGF-ß pathway signaling. Treatment of fibroblasts with an inhibitor of miR-17-5p reversed the contraction and migration phenotypes induced by epithelial-derived exosomes. Exosomal miR-17-5p was confirmed to function as a key regulator of the phenotypic transformation of fibroblasts. In conclusion, we demonstrated that Arecoline triggers aberrant epithelium-fibroblast crosstalk and identified that epithelial cell-derived miR-17-5p mediates fibroblast differentiation through the classical TGF-ß fibrotic pathway, which provided a new perspective and strategy for the diagnosis and treatment of OSF.
Assuntos
Arecolina , Células Epiteliais , Exossomos , Fibroblastos , MicroRNAs , Fibrose Oral Submucosa , Receptor do Fator de Crescimento Transformador beta Tipo I , MicroRNAs/metabolismo , Fibrose Oral Submucosa/metabolismo , Fibrose Oral Submucosa/patologia , Humanos , Fibroblastos/metabolismo , Arecolina/farmacologia , Células Epiteliais/metabolismo , Exossomos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteína Smad7/metabolismo , Diferenciação Celular , Transdução de Sinais , Movimento Celular , Ubiquitina-Proteína Ligases/metabolismo , Areca/efeitos adversosRESUMO
INTRODUCTION: Colorectal cancer (CRC) is the second common cancer and the fourth major reason of cancer death worldwide. Dysregulation of intracellular pathways, such as TGF-ß/SMAD signaling, contributes to CRC development. MicroRNAs (miRNAs) are post-transcriptional regulators that are involved in CRC pathogenesis. Here, we aimed to investigate the effect of miR-3613-3p on the TGF-ß /SMAD signaling pathway in CRC. METHODS & RESULTS: Bioinformatics analysis suggested that miR-3613-3p is a regulator of TGF-Β signaling downstream genes. Then, miR-3613-3p overexpression was followed by downregulation of TGF-ßR1, TGF-ßR2, and SMAD2 expression levels, detected by RT-qPCR. Additionally, dual luciferase assay supported the direct interaction of miR-3613-3p with 3'UTR sequences of TGF-ßR1 and TGF-ßR2 genes. Furthermore, reduced SMAD3 protein level following the miR-3613-3p overexpression verified its suppressive effect against TGF-ß signaling in HCT-116 cells, detected by western blot analysis. Finally, miR-3613-3p overexpression induced sub-G1 arrest in HCT116 cells, detected by flow cytometry, and promoted downregulation of cyclin D1 protein expression, which was detected by western blotting analysis. CONCLUSION: Our findings indicated that miR-3613-3p plays an important role in CRC by targeting the TGF-ß/SMAD signaling pathway and could be considered as a new candidate for further therapy investigations.
Assuntos
Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proliferação de Células/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
Transforming growth factor beta (TGF-ß) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-ß receptor type I (Mx1;TßRICA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TßRICA mice using RNA sequencing analysis. A total of 285 genes, including 87 upregulated and 198 downregulated genes, were differentially expressed. According to the KEGG analysis, some genes were involved in osteoclast differentiation (Fcgr4, Lilrb4a), B cell receptor signaling (Cd72, Lilrb4a), and neutrophil extracellular trap formation (Hdac7, Padi4). Lilrb4 is related to osteoclast inhibition protein, whereas Hdac7 is a Runx2 corepressor that regulates osteoblast differentiation. Silencing Lilrb4 increased the number of osteoclasts and osteoclast marker genes. The knocking down of Hdac7 increased alkaline phosphatase activity, mineralization, and osteoblast marker genes. Therefore, our present study may provide an innovative idea for potential therapeutic targets and pathways in TßRI-associated bone loss.
Assuntos
Remodelação Óssea , Osteoclastos , Animais , Camundongos , Remodelação Óssea/genética , Osteoclastos/metabolismo , Osteoclastos/citologia , Osteoblastos/metabolismo , Regulação da Expressão Gênica , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Diferenciação Celular/genética , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Perfilação da Expressão GênicaRESUMO
Myocardial fibrosis is a pathological, physiological change that results from alterations, such as inflammation and metabolic dysfunction, after myocardial infarction (MI). Excessive fibrosis can cause cardiac dysfunction, ventricular remodeling, and heart failure. Caffeic acid (CA), a natural polyphenolic acid in various foods, has cardioprotective effects. This study aimed to explore whether CA exerts a cardioprotective effect to inhibit myocardial fibrosis post-MI and elucidate the underlying mechanisms. Histological observations indicated that CA ameliorated ventricular remodeling induced by left anterior descending coronary artery ligation in MI mice and partially restored cardiac function. CA selectively targeted transforming growth factor-ß receptor 1 (TGFBR1) and inhibited TGFBR1-Smad2/3 signaling, reducing collagen deposition in the infarcted area of MI mice hearts. Furthermore, cell counting (CCK-8) assay, 5-ethynyl-2'-deoxyuridine assay, and western blotting revealed that CA dose-dependently decreased the proliferation, collagen synthesis, and activation of the TGFBR1-Smad2/3 pathway in primary cardiac fibroblasts (CFs) stimulated by TGF-ß1 in vitro. Notably, TGFBR1 overexpression in CFs partially counteracted the inhibitory effects of CA. These findings suggest that CA effectively mitigates myocardial fibrosis and enhances cardiac function following MI and that this effect may be associated with the direct targeting of TGFBR1 by CA.
Assuntos
Ácidos Cafeicos , Fibrose , Camundongos Endogâmicos C57BL , Infarto do Miocárdio , Miocárdio , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Animais , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Masculino , Ácidos Cafeicos/farmacologia , Ácidos Cafeicos/uso terapêutico , Miocárdio/patologia , Miocárdio/metabolismo , Camundongos , Remodelação Ventricular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Colágeno/metabolismo , Proteína Smad2/metabolismo , Células CultivadasRESUMO
The role of mitochondria peptides in the spreading of glioblastoma remains poorly understood. In this study, we investigated the mechanism underlying intracranial glioblastoma progression. Our findings demonstrate that the mitochondria-derived peptide, humanin, plays a significant role in enhancing glioblastoma progression through the intratumoral activation of the integrin alpha V (ITGAV)-TGF beta (TGFß) signaling axis. In glioblastoma tissues, humanin showed a significant upregulation in the tumor area compared to the corresponding normal region. Utilizing multiple in vitro pharmacological and genetic approaches, we observed that humanin activates the ITGAV pathway, leading to cellular attachment and filopodia formation. This process aids the subsequent migration and invasion of attached glioblastoma cells through intracellular TGFßR signaling activation. In addition, our in vivo orthotopic glioblastoma model provides further support for the pro-tumoral function of humanin. We observed a correlation between poor survival and aggressive invasiveness in the humanin-treated group, with noticeable tumor protrusions and induced angiogenesis compared to the control. Intriguingly, the in vivo effect of humanin on glioblastoma was significantly reduced by the treatment of TGFBR1 inhibitor. To strengthen these findings, public database analysis revealed a significant association between genes in the ITGAV-TGFßR axis and poor prognosis in glioblastoma patients. These results collectively highlight humanin as a pro-tumoral factor, making it a promising biological target for treating glioblastoma.
Assuntos
Progressão da Doença , Glioblastoma , Integrina alfaV , Transdução de Sinais , Fator de Crescimento Transformador beta , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Humanos , Fator de Crescimento Transformador beta/metabolismo , Animais , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Integrina alfaV/metabolismo , Integrina alfaV/genética , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Movimento Celular/efeitos dos fármacos , Camundongos Nus , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Invasividade Neoplásica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Renal fibrosis is a representative pathological feature of various chronic kidney diseases, and efficient treatment is needed. Interstitial myofibroblasts are a key driver of kidney fibrosis, which is dependent on the binding of TGF-ß1 to type I TGF-ß receptor (TßRI) and TGF-ß1-related signaling pathways. Therefore, attenuating TGF-ß1 activity by competing with TGF-ß1 in myofibroblasts is an ideal strategy for treating kidney fibrosis. Recently, a novel TßRI-mimicking peptide RIPΔ demonstrated a high affinity for TGF-ß1. Thus, it could be speculated that RIPΔ may be used for anti-fibrosis therapy. Platelet-derived growth factor ß receptor (PDGFßR) is highly expressed in fibrotic kidney. In this study, we found that target peptide Z-RIPΔ, which is RIPΔ modified with PDGFßR-specific affibody ZPDGFßR, was specifically and highly taken up by TGF-ß1-activated NIH3T3 fibroblasts. Moreover, Z-RIPΔ effectively inhibited the myofibroblast proliferation, migration and fibrosis response in vitro. In vivo and ex vivo experiments showed that Z-RIPΔ specifically targeted fibrotic kidney, improved the damaged renal function, and ameliorated kidney histopathology and renal fibrosis in UUO mice. Mechanistic studies showed that Z-RIPΔ hold the stronger inhibition of the TGF-ß1/Smad and TGF-ß1/p38 pathways than unmodified RIPΔ in vitro and in vivo. Furthermore, systemic administration of Z-RIPΔ to UUO mice led to minimal toxicity to major organs. Taken together, RIPΔ modified with ZPDGFßR increased its therapeutic efficacy and reduced its systemic toxicity, making it a potential candidate for targeted therapy for kidney fibrosis.
Assuntos
Fibrose , Rim , Camundongos Endogâmicos C57BL , Proteínas Smad , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Fibrose/tratamento farmacológico , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Células NIH 3T3 , Masculino , Proteínas Smad/metabolismo , Transdução de Sinais/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Peptídeos/uso terapêutico , Peptídeos/farmacologia , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Nefropatias/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Humanos , Modelos Animais de Doenças , Proliferação de Células/efeitos dos fármacosRESUMO
Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.
Assuntos
Prótese Vascular , Colágeno , Células-Tronco Pluripotentes Induzidas , Receptor do Fator de Crescimento Transformador beta Tipo I , Animais , Humanos , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Colágeno/metabolismo , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ratos Nus , Modelos Animais de Doenças , Ratos , Bioengenharia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Edição de Genes , Síndrome de Loeys-Dietz/genética , Síndrome de Loeys-Dietz/patologia , MasculinoRESUMO
Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor ß (TGFß), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFß3 in regulating TX generation in human ovarian follicular GCs. TGFß3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFß3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFßRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFß3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFß3. Taken together, the results presented here first demonstrated that FF TGFß3 levels differ significantly in IVF patients' large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFß3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFßR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFß3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation.
Assuntos
Ciclo-Oxigenase 2 , Líquido Folicular , Células da Granulosa , Transdução de Sinais , Proteína Smad2 , Proteína Smad3 , Fator de Crescimento Transformador beta3 , Humanos , Feminino , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/genética , Células da Granulosa/metabolismo , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proteína Smad3/metabolismo , Proteína Smad3/genética , Líquido Folicular/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta3/genética , Adulto , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Folículo Ovariano/metabolismo , Oócitos/metabolismo , Células CultivadasRESUMO
Polymorphism of genes of transforming growth factor TGFB and its receptors (TGFBRI, TGFBRII, and TGFBRIIII) in patients with primary open-angle glaucoma was analyzed. The frequency of the TGFBRII CC genotype in patients is increased relative to the control group (OR=6.10, p=0.0028). Heterozygosity in this polymorphic position is reduced (OR=0.18, p=0.0052). As the effects of TGF-ß is mediated through its receptors, we analyzed complex of polymorphic variants of the studied loci in the genome of patients. Two protective complexes consisting only of receptor genes were identified: TGFBRI TT:TGFBRII CG (OR=0.10, p=0.02) and TGFBRII CG:TGFBRIII CG (OR=0.09, p=0.01). The study showed an association of TGFBRII polymorphism with primary open-angle glaucoma and the need to study functionally related genes in the development of the disease, which should contribute to its early diagnosis and prevention.