RESUMO
In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-ß family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.
Assuntos
Proteínas de Drosophila , Drosophila , Fator de Crescimento Transformador beta , Animais , Membrana Celular/metabolismo , Cães , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Ligantes , Células Madin Darby de Rim Canino , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor-beta (TGFß) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFß is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFß induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFß-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFß signaling, TGFß stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.
Assuntos
Antígeno B7-H1/biossíntese , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células 3T3 , Animais , Antígeno B7-H1/genética , Vesículas Extracelulares/patologia , Fibroblastos/patologia , Fibrose , Regulação da Expressão Gênica , Humanos , Camundongos , Fator de Crescimento Transformador beta/genéticaRESUMO
In the current work we show that the profibrotic actions of TGF-ß are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-ß required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-ß. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-ß. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-ß-dependent manner.
Assuntos
Fibroblastos/patologia , Regulação da Expressão Gênica , Glutaminase/metabolismo , Fibrose Pulmonar/patologia , Sirtuínas/metabolismo , Fator de Crescimento Transformador beta/toxicidade , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Movimento Celular , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Glutaminase/genética , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Sirtuínas/genética , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMO
Pathogenic fibrotic diseases, including idiopathic pulmonary fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFß) and insulin-like growth factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFß-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFß in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF-1's C1 promoter as mediating the increase in IGF-1 transcription by TGFß in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFß-dependent Igf-1 expression in myofibroblasts; (4) demonstrates IGF-1R activation is essential to support TGFß-driven profibrotic myofibroblast functions and excessive wound healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1's role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Evidence is provided that the fibroproliferative actions of TGF-ß are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF-ß signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF-ß-stimulated FASN expression is independent of Smad2/3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF-ß-driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin-treated mice, inhibition of FASN was examined in a murine-treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.-Jung, M.-Y., Kang, J.-H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez-Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF-ß signaling.
Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Bleomicina/toxicidade , Linhagem Celular , Ácido Graxo Sintase Tipo I/genética , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/etiologia , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
TGF-ß plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-ß in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-ß. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is shown to impair TGF-ß-driven fibrogenic processes, including cell proliferation and production of profibrotic mediators. These findings provide new perspectives on the interrelation of metabolism and profibrotic TGF-ß signaling and present opportunities for potential therapeutic intervention.-Andrianifahanana, M., Hernandez, D. M., Yin, X., Kang, J.-H., Jung, M.-Y., Wang, Y., Yi, E. S., Roden, A. C., Limper, A. H., Leof, E. B. Profibrotic up-regulation of glucose transporter 1 by TGF-ß involves activation of MEK and mammalian target of rapamycin complex 2 pathways.
Assuntos
Proliferação de Células/fisiologia , Transportador de Glucose Tipo 1/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Sirolimo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Fibrose/metabolismo , Pulmão/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
PURPOSE: To determine if adventitial transplantation of human adipose tissue-derived mesenchymal stem cells (MSCs) to the outflow vein of B6.Cg-Foxn1(nu)/J mice with arteriovenous fistula (AVF) at the time of creation would reduce monocyte chemoattractant protein-1 (Mcp-1) gene expression and venous neointimal hyperplasia. The second aim was to track transplanted zirconium 89 ((89)Zr)-labeled MSCs serially with positron emission tomography (PET) for 21 days. MATERIALS AND METHODS: All animal experiments were performed according to protocols approved by the institutional animal care and use committee. Fifty B6.Cg-Foxn1(nu)/J mice were used to accomplish the study aims. Green fluorescent protein was used to stably label 2.5 × 10(5) MSCs, which were injected into the adventitia of the outflow vein at the time of AVF creation in the MSC group. Eleven mice died after AVF placement. Animals were sacrificed on day 7 after AVF placement for real-time polymerase chain reaction (n = 6 for MSC and control groups) and histomorphometric (n = 6 for MSC and control groups) analyses and on day 21 for histomorphometric analysis only (n = 6 for MSC and control groups). In a separate group of experiments (n = 3), animals with transplanted (89)Zr-labeled MSCs were serially imaged with PET for 3 weeks. Multiple comparisons were performed with two-way analysis of variance, followed by the Student t test with post hoc Bonferroni correction. RESULTS: In vessels with transplanted MSCs compared with control vessels, there was a significant decrease in Mcp-1 gene expression (day 7: mean reduction, 62%; P = .029), with a significant increase in the mean lumen vessel area (day 7: mean increase, 176% [P = .013]; day 21: mean increase, 415% [P = .011]). Moreover, this was accompanied by a significant decrease in Ki-67 index (proliferation on day 7: mean reduction, 81% [P = .0003]; proliferation on day 21: mean reduction, 60%, [P = .016]). Prolonged retention of MSCs at the adventitia was evidenced by serial PET images of (89)Zr-labeled cells. CONCLUSION: Adventitial transplantation of MSCs decreases Mcp-1 gene expression, accompanied by a reduction in venous neointimal hyperplasia.
Assuntos
Derivação Arteriovenosa Cirúrgica/efeitos adversos , Transplante de Células-Tronco Mesenquimais , Neointima/patologia , Tecido Adiposo/citologia , Animais , Humanos , Hiperplasia/patologia , Hiperplasia/prevenção & controle , Marcação In Situ das Extremidades Cortadas , Camundongos , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: To determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia. MATERIALS AND METHODS: Chronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 10(6) plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses. RESULTS: By day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels. CONCLUSIONS: A second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21.
Assuntos
Derivação Arteriovenosa Cirúrgica/efeitos adversos , Vetores Genéticos , Oclusão de Enxerto Vascular/prevenção & controle , Veias Jugulares/cirurgia , Lentivirus/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Insuficiência Renal Crônica/terapia , Fator A de Crescimento do Endotélio Vascular/genética , Actinas/metabolismo , Túnica Adventícia/metabolismo , Animais , Apoptose , Artérias Carótidas/cirurgia , Proliferação de Células , Modelos Animais de Doenças , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Veias Jugulares/metabolismo , Veias Jugulares/patologia , Lentivirus/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Neointima , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Remodelação VascularRESUMO
OBJECTIVE: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive. APPROACH AND RESULTS: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis. CONCLUSIONS: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.
Assuntos
Cardiomiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Isquemia Miocárdica/metabolismo , Neuropilina-1/metabolismo , Animais , Homeostase , Camundongos Knockout , Proteínas dos Microfilamentos , Mitocôndrias Cardíacas/metabolismo , Proteínas Musculares , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , PPAR gama/metabolismo , Receptor Cross-Talk , Receptor Notch1/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. MATERIALS AND METHODS: AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. RESULTS: Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. CONCLUSION: These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation.
Assuntos
Derivação Arteriovenosa Cirúrgica/efeitos adversos , Angiopatias Diabéticas/enzimologia , Fibroblastos/enzimologia , Glucose/metabolismo , Oclusão de Enxerto Vascular/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Neovascularização Patológica , Diálise Renal , Animais , Hipóxia Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Meios de Cultivo Condicionados/metabolismo , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.
Assuntos
Túnica Adventícia/cirurgia , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Oclusão de Enxerto Vascular/prevenção & controle , Veias Jugulares/cirurgia , Lentivirus/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Túnica Adventícia/metabolismo , Túnica Adventícia/patologia , Animais , Apoptose , Artérias Carótidas/cirurgia , Caspase 3/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Constrição Patológica , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Hiperplasia , Veias Jugulares/metabolismo , Veias Jugulares/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neointima , Nefrectomia , RNA Interferente Pequeno/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Transforming growth factor ß (TGFß) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFß induced ErbB ligands and the physiological significance of inhibiting multiple TGFß-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFß-stimulated ErbB ligand up-regulation, TGFß-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFß/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFß signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFß.
Assuntos
Receptores ErbB/metabolismo , Fibrose Pulmonar/metabolismo , Receptor ErbB-2/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Benzamidas/administração & dosagem , Benzamidas/uso terapêutico , Bleomicina/toxicidade , Linhagem Celular , Interações Medicamentosas , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Retroalimentação Fisiológica , Mesilato de Imatinib , Lapatinib , Pulmão/fisiopatologia , Camundongos , Miofibroblastos/metabolismo , Comunicação Parácrina , Piperazinas/administração & dosagem , Piperazinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Troca Gasosa Pulmonar , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Quinazolinas/administração & dosagem , Quinazolinas/uso terapêutico , Regulação para CimaRESUMO
Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity.
Assuntos
Derivação Arteriovenosa Cirúrgica/efeitos adversos , Oclusão de Enxerto Vascular/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Veias Jugulares/efeitos dos fármacos , Diálise Renal , Sinvastatina/farmacologia , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Artérias Carótidas/cirurgia , Caspase 3/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Constrição Patológica , Modelos Animais de Doenças , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Hiperplasia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Veias Jugulares/metabolismo , Veias Jugulares/patologia , Veias Jugulares/cirurgia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Células NIH 3T3 , Neointima , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
TGF-ß modulates numerous diverse cellular phenotypes including growth arrest in epithelial cells and proliferation in fibroblasts. Although the Smad pathway is fundamental for the majority of these responses, recent evidence indicates that non-Smad pathways may also have a critical role. Here we report a novel mechanism whereby the nonreceptor tyrosine focal adhesion kinase (FAK) functions as an adaptor necessary for cell type-specific responses to TGF-ß. We show that in contrast to Smad actions, non-Smad pathways, including c-Abl, PAK2, and Akt, display an obligate requirement for FAK. Interestingly, this occurs in Src null SYF cells and is independent of FAK tyrosine phosphorylation, kinase activity, and/or proline-rich sequences in the C-terminal FAT domain. FAK binds the phosphatidylinositol 3-kinase (PI3K) p85 regulatory subunit following TGF-ß treatment in a subset of fibroblasts but not epithelial cells and has an obligate role in TGF-ß-stimulated anchorage-independent growth and migration. Together, these results uncover a new scaffolding role for FAK as the most upstream component regulating the profibrogenic action of TGF-ß and suggest that inhibiting this interaction may be useful in treating a number of fibrotic diseases.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Cães , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Quinase 1 de Adesão Focal/genética , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Smad , Células Swiss 3T3 , Fator de Crescimento Transformador beta/genéticaRESUMO
The transforming growth factor-beta (TGF-ß) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-ß1 (LAP-TGF-ß1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-ß stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-ß1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.
Assuntos
Fibroblastos/metabolismo , Mioblastos/metabolismo , Neuropilina-1/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad Reguladas por Receptor/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular Transformada , Fibroblastos/citologia , Regulação da Expressão Gênica/fisiologia , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Mioblastos/citologia , Neuropilina-1/genética , Fosforilação/fisiologia , Proteínas Smad Reguladas por Receptor/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) remains an intractably fatal disorder, despite the recent advent of anti-fibrotic medication. Successful treatment of IPF, like many chronic diseases, may benefit from the concurrent use of multiple agents that exhibit synergistic benefit. In this light, phosphodiesterase type 5 inhibitors (PDE5-Is), have been studied in IPF primarily for their established pulmonary vascular effects. However, recent data suggest certain PDE5-Is, particularly vardenafil, may also reduce transforming growth factor beta 1 (TGF-ß1) activation and extracellular matrix (ECM) accumulation, making them a potential target for therapy for IPF. We evaluated fibroblast TGF-ß1-driven extracellular matrix (ECM) generation and signaling as well as epithelial mesenchymal transformation (EMT) with pretreatment using the PDE5-I vardenafil. In addition, combinations of vardenafil and nintedanib were evaluated for synergistic suppression of EMC using a fibronectin enzyme-linked immunosorbent assay (ELISA). Finally, the effects of vardenafil on fibrosis were investigated in a bleomycin mouse model. Our findings demonstrate that vardenafil suppresses ECM generation alone and also exhibits significant synergistic suppression of ECM in combination with nintedanib in vitro. Interestingly, vardenafil was shown to improve fibrosis markers and increase survival in bleomycin-treated mice. Vardenafil may represent a potential treatment for IPF alone or in combination with nintedanib. However, additional studies will be required.
Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Indóis/uso terapêutico , Dicloridrato de Vardenafila/uso terapêutico , Animais , Bleomicina , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Indóis/farmacologia , Pulmão/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Análise de Sobrevida , Fator de Crescimento Transformador beta1/metabolismo , Dicloridrato de Vardenafila/farmacologiaRESUMO
Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.
Assuntos
Nefropatias/metabolismo , Rim/metabolismo , Rim/patologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Benzamidas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibroblastos/patologia , Fibrose , Mesilato de Imatinib , Nefropatias/etiologia , Nefropatias/patologia , Masculino , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Obstrução Ureteral/complicaçõesRESUMO
Transforming growth factor beta (TGFbeta), a multifunctional cytokine associated with vascular injury, is a potent inhibitor of cell proliferation. The current results demonstrate that the TGFbeta-induced growth arrest of vascular smooth muscle cells (VSMCs) is associated with cyclin A downregulation. TGFbeta represses the cyclin A gene through a cyclic AMP (cAMP) response element, which complexes with the cAMP response element binding protein (CREB). The CREB-cyclin A promoter interaction is hindered by TGFbeta, preceded by a TGFbeta receptor-dependent CREB phosphorylation. Induction of CREB phosphorylation with forskolin or 6bnz-cAMP mimics TGFbeta's inhibitory effect on cyclin A expression. Conversely, inhibition of CREB phosphorylation with a CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A) upregulated cyclin A gene expression. Furthermore, the CREB-S133A mutant abolished TGFbeta-induced CREB phosphorylation, cyclin A downregulation, and growth inhibition. Since we have previously shown that the novel PKC isoform protein kinase C delta (PKCdelta) is activated by TGFbeta in VSMCs, we tested the role of this kinase in CREB phosphorylation and cyclin A downregulation. Inhibition of PKCdelta by a dominant-negative mutant or by targeted gene deletion blocked TGFbeta-induced CREB phosphorylation and cyclin A downregulation. Taken together, our data indicate that phosphorylation of CREB stimulated by TGFbeta is a critical step leading to the inhibition of cyclin A expression and, thus, VSMC proliferation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclina A/metabolismo , Regulação para Baixo/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Linhagem Celular , Ciclina A/genética , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteína Quinase C-delta/deficiência , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Ratos , Elementos de RespostaRESUMO
PURPOSE: Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts that have become myofibroblasts (ie, alpha-smooth muscle actin [SMA]-positive cells) and migrate to the neointima. There is increased expression of hypoxia-inducible factor (HIF)-1alpha in venous neointimal hyperplasia formation in experimental animal models and clinical samples. It was hypothesized that, under hypoxic stimulus (ie, HIF-1alpha), fibroblasts will convert to myofibroblasts through a matrix metalloproteinase (MMP)-2-mediated pathway. MATERIALS AND METHODS: Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1alpha, alpha-SMA, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for alpha-SMA, collagen, and fibronectin was performed. RESULTS: At all time points, there was significantly increased expression of HIF-1alpha in the hypoxic fibroblasts compared with normoxic fibroblasts (P < .05). There was significantly increased expression of alpha-SMA at all time points, which peaked by 48 hours in hypoxic fibroblasts compared with normoxic fibroblasts (P < .05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in TIMP-1 by 48-72 hours by hypoxic fibroblasts (P < .05). By 72 hours, there was significant increase in TIMP-2 expression (P < .05). Immunohistochemical analysis demonstrated increased expression of alpha-SMA, collagen, and fibronectin as the duration of hypoxia increased. CONCLUSIONS: Under hypoxic conditions, fibroblasts will convert to myofibroblasts through an MMP-2-mediated pathway, which may provide insight into the mechanism of venous neointimal hyperplasia.
Assuntos
Fibroblastos/citologia , Fibroblastos/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular , Camundongos , Diálise Renal/métodos , Túnica Íntima/fisiopatologiaRESUMO
Transforming growth factor (TGF)-beta receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGFbeta type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGFbeta-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment.