RESUMO
Endogenous bone marrow-derived mesenchymal stem cells are mobilized to peripheral blood and injured tissues in response to changes in the expression of various growth factors and cytokines in the injured tissues, including substance P (SP), transforming growth factor-beta (TGF-ß), and stromal cell-derived factor-1 (SDF-1). SP, TGF-ß, and SDF-1 are all known to induce the migration of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is not yet clear how these stimuli influence or interact with each other during BM-MSC mobilization. This study used mouse bone marrow-derived mesenchymal stem cell-like ST2 cells and human BM-MSCs to evaluate whether SP, TGF-ß, and SDF-1 mutually regulate their respective effects on the mobilization of BM-MSCs. SP pretreatment of ST2 and BM-MSCs impaired their response to TGF-ß while the introduction of SP receptor antagonist restored the mobilization of ST2 and BM-MSCs in response to TGF-ß. TGF-ß pretreatment did not affect the migration of ST2 and BM-MSCs in response to SP, but downregulated their migration in response to SDF-1. SP pretreatment modulated the activation of TGF-ß noncanonical pathways in ST2 cells and BM-MSCs, but not canonical pathways. These results suggest that the migration of mesenchymal stem cells is regulated by complex functional interactions between SP, TGF-ß, and SDF-1. Thus, understanding the complex functional interactions of these chemotactic stimuli would contribute to ensuring the development of safe and effective combination treatments for the mobilization of BM-MSCs.
Assuntos
Células da Medula Óssea/imunologia , Quimiotaxia/imunologia , Células-Tronco Mesenquimais/imunologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Quimiocina CXCL12/imunologia , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Fator de Crescimento Transformador beta/imunologiaRESUMO
Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.
Assuntos
Proteína Morfogenética Óssea 4/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias/etiologia , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Proteína Smad4/genética , Proteína Smad4/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transforming growth factor-beta (TGF-ß) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-ß mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-ß are unclear. In this study, we showed that TGF-ß activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-ß type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-ß-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-ß were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-ß-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-ß. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-ß was mediated through N-cadherin and noncanonical TGF-ß signals.
Assuntos
Antígenos CD/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Antígenos CD/genética , Células da Medula Óssea/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neuropeptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/agonistas , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Preservation of the pancreatic ß-cell population is required for the development of therapies for diabetes, which is caused by a decrease in ß-cells. Here, we demonstrate the antidiabetic effects of substance P (SP) in type 1 diabetes (T1D) mice induced with streptozotocin. SP enhanced the compensatory proliferation of ß-cells in order to restore ß-cells in response to acute injury induced by a single high-dose of streptozotocin. However, SP affected neither the basal proliferation of ß-cells nor their apoptosis. In vitro studies by using the INS-1 pancreatic ß-cell line showed that SP mediated the increase in the proliferation of ß-cells via the activation of Akt. Chronic systemic treatment with SP restored the mass of ß-cells and inhibited insulitis in T1D mice induced with multiple low-doses of streptozotocin. Therefore, systemic treatment with SP may be a promising therapeutic strategy for treating diabetes in patients with loss of functional ß-cells.
Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/prevenção & controle , Células Secretoras de Insulina/efeitos dos fármacos , Pancreatite/prevenção & controle , Substância P/farmacologia , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Pancreatite/induzido quimicamente , Pancreatite/patologia , Estreptozocina/administração & dosagem , Relação Estrutura-AtividadeRESUMO
Substance P (SP) is known to induce the mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) and thus participates in wound repair. However, the cellular and molecular mechanisms responsible for the SP-mediated migration of BM-MSCs were not fully understood. In the present study, we studied the molecular mechanisms that mediate the migration of the BM-derived MSC-like cell line ST2 in response to SP. Using a migration assay and western blot analysis, we noted that SP induced the chemotactic migration of ST2 cells through the intrinsic activation of extracellular signal-regulated kinases (ERKs) and protein kinase B (Akt), the phosphorylated expression levels of which were increased. We noted that Src is involved in the SP-mediated migration of ST2 cells and that focal adhesion kinase (FAK) was activated in the ST2 cells following SP treatment. Membrane ruffling increased in the ST2 cells after SP treatment, as was clearly demonstrated by immunocytochemical analysis. Importantly, using a blocking antibody against N-cadherin (GC-4), we studied cell migration and noted that SP mediated the migration of the ST2 cells through N-cadherin. The present study thus advanced our understanding of the mechanisms through which SP induces BM-MSC migration.
Assuntos
Células-Tronco Mesenquimais/citologia , Substância P/metabolismo , Animais , Caderinas/metabolismo , Movimento Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Dermal fibroblasts play essential roles in wound healing. However, they lose their normal regenerative functions under certain pathologic conditions such as in chronic diabetic wounds. Here, we show that substance P (SP) rescues the malfunctions of dermal fibroblasts in diabetes. SP increased the proliferation of diabetic dermal fibroblasts dose-dependently, although the effect was lower compared to the SP-stimulated proliferation of normal dermal fibroblasts. In contrast to normal dermal fibroblasts, SP increased the expression level of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) in diabetic dermal fibroblast hence, rescuing their angiogenic potential. The cellular characteristics of diabetic dermal fibroblasts modulated by SP would be able to accelerate the wound healing process through faster wound contraction and improved angiogenesis in diabetic chronic wounds. Moreover, SP pretreatment into dermal fibroblasts isolated from diabetic patients would be a promising strategy to develop autologous cell therapy for treating diabetic chronic wounds.
RESUMO
Impaired angiogenesis is a common pathological characteristic of chronic wounds. Therefore, the regulation of angiogenesis is important for proper tissue repair. It was reported that substance P (SP) accelerates wound healing in a skin injury model. SP is degraded by neutral endopeptidase (NEP). Our study shows that systemic co-treatment of SP and thiorphan, an inhibitor of NEP synergically increased the number of α-smooth muscle actin positive-blood vessels in skin wounds. However, there was no synergic improvement in wound contraction and extracellular matrix deposition. Therefore, inhibition of endogenous NEP activity by thiorphan treatment might modulate the effects of SP treatment specifically on accelerating angiogenesis during wound healing. However, the molecular mechanism(s) of the synergic increase in angiogenesis by SP and thiorphan treatment is still unknown.
RESUMO
The rescue of glucose tolerance and insulinsensitivity in peripheral tissues, including adipose tissue, is essential in therapeutic strategies for diabetes. The present study demonstrated that substance P (SP) increases the accumulation of lipids in 3T3L1 cells during their differentiation into adipocytes in response to a high concentration of glucose. SP reciprocally regulated the activities of AMPactivated protein kinase (AMPK) and Akt: SP enhanced the activation of AMPK, although the activity of Akt was downregulated. Notably, SP induced an increase in the expression level of glucose transporter 4 in the 3T3L1 adipocytes. Therefore, it is possible that SP leads to an increase in glucose uptake and the accumulation of lipids in adipocytes, and may contribute towards the rescue of insulinsensitivity in diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Substância P/farmacologia , Células 3T3-L1 , Animais , Regulação para Baixo/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Due to the therapeutic characteristics of bone marrow (BM)-derived mesenchymal stem cells (MSCs), clinical trials are testing the use of autologous or allogeneic MSCs for the treatment of several conditions. These therapies require large numbers of MSCs and numerous studies are attempting to find substances that could enhance the egression of endogenous MSCs from the BM into the periphery and increase their proliferation in vivo and in vitro. It has been reported that substance P (SP) has the potential to increase the expansion of MSCs in vivo and to induce their mobilization from the BM into the periphery. The aim of the present study was to investigate the effects of SP on the migration and proliferation potential of two BM-derived MSC-like cell lines, ST2 and OP9. SP was found to induce the migration potential of ST2 cells in vitro. Furthermore, SP increased the proliferation of the MSCs cell line, OP9 cell line. Cyclin D1 expression was observed to increase in the OP9 cells, indicating the activation of the cell cycle in response to SP. The upstream signals involved in these phenomena have yet to be elucidated, although previous studies have proposed the activation of the extracellular signal-regulated kinase-1/2 and Wingless/ß-catenin pathways as possible mediators of the cellular proliferation of human MSCs in response to SP. The present results therefore suggest that SP would facilitate the obtainment of higher numbers of endogenous MSCs from patients or donors and/or shorten the process of in vitro expansion that could cause the MSCs to undergo changes in their innate therapeutic characteristics prior to their use in therapy.
RESUMO
Epithelial-mesenchymal transition (EMT) of mammary epithelial cells is important in both normal morphogenesis of mammary glands and metastasis of breast cancer. Cadherin switching from E-cadherin to N-cadherin plays important roles in EMT. We found that cadherin switching is important in bone morphogenetic protein 4 (BMP4)-induced EMT in MCF-10A cells. BMP4 increased the phosphorylation of SMAD proteins in MCF-10A cells. Canonical BMP4 signaling decreased the expression of E-cadherin and disrupted the polarity of the tight junction protein ZO-1 in MCF-10A cells. However, the expression of N-cadherin and SNAI2 was up-regulated in BMP4-treated MCF-10A cells. MCF-10A cells that expressed N-cadherin migrated into type I collagen gels in response to BMP4 when evaluated using three-dimensional culture assays. Thus, active canonical BMP4 signaling is important for the migration and EMT of mammary epithelial cells. Moreover, the decrease in E-cadherin and/or increase in N-cadherin may be required for BMP4-induced migration and EMT.