RESUMO
We have assessed the molecular role of Rutin and rutin-Zn(II) complex on osteoblast differentiation and mineralization in human dental pulp cells and zebrafish model. The biocompatibility of the rutin-Zn(II) complex was determined using MTT and chick embryotoxicity assays. Alizarin red staining and ALP measurements were performed to study the osteogenic role of Rutin and rutin-Zn(II) complex at the cellular level in hDPSCs. At molecular level, following rutin and rutin-Zn(II) exposure, the mRNA expression profile of osteoblast markers such Runx2, type 1 col, OC, and ON were investigated. In addition to this, the expression of negative regulators of osteoblast development such Smad7, Smurf1, and HDAC7 waere studied by Real time RT-PCR analysis. The osteogenic role of prepared complex under in vivo was studied by an in-house zebrafish scale model followed by osteoblast differentiation markers expression profiling and Ca:P level measurement by ICP-MS. Rutin and the rutin-Zn(II) complex were found to be non-toxic till 10 µM and increased the expression of osteoblast differentiation marker genes. It also enhanced calcium deposition in both in vitro and in vivo models. Osteogenic property of rutin-Zn(II) in hDPSCs was found be mediated by Smad7, Smurf1, and HDAC7 and enhancing Runx2 expression. Our study warrants the possible use of rutin-Zn(II) as naïve agent or in combination with other bone scaffolding systems/materials for bone tissue engineering applications.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Osteogênese/efeitos dos fármacos , Rutina/química , Zinco/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Polpa Dentária/citologia , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Proteína Smad7/genética , Proteína Smad7/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Smad7 is the main negative regulatory protein in the transforming growth factor-ß (TGF-ß) downstream signaling pathway, which plays an important role in diabetic nephropathy (DN) and may be related to the ubiquitin proteasome pathway (UPP). AIM: We investigated the role of UPP in regulating TGF-ß/SMAD signaling and explored the therapeutic effect of the ubiquitin proteasome inhibitor MG132 on DN. METHODS: Wistar rats were randomly divided into a diabetes group and a normal control group. Rats in the diabetes group were injected intraperitoneally with streptozotocin. Diabetic rats were then randomly divided into a diabetic nephropathy group (DN group), an MG132 high concentration (MH) group, and an MG132 low concentration (ML) group. After 8 weeks of treatment, 24-hour urinary microalbumin (UAlb), urinary protein/urinary creatinine (Up/Ucr) values, ALT, AST, Bcr, kidney damage, TGF-ß, Smad7, fibronectin (FN), and Smurf2 were detected. RESULTS: The body mass and Smad7 protein expression decreased in DN group, but kidney weight, kidney weight index, UAlb, Up/Ucr, FN and Smurf2 mRNA expression, and TGF-ß protein expression increased. However, these changes diminished following treatment with MG132, and a more pronounced effect was evident in MH group compared to ML group. CONCLUSION: MG132 alleviates kidney damage by inhibiting Smad7 ubiquitin degradation and TGF-ß activation in DN.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Rim/efeitos dos fármacos , Leupeptinas/uso terapêutico , Inibidores de Proteassoma/uso terapêutico , Proteína Smad7/metabolismo , Albuminúria/prevenção & controle , Animais , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Fibronectinas/antagonistas & inibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Leupeptinas/administração & dosagem , Masculino , Tamanho do Órgão/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/administração & dosagem , Proteólise/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteína Smad7/antagonistas & inibidores , Proteína Smad7/genética , Estreptozocina , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.
