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1.
J Pediatr Orthop ; 42(6): e590-e595, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35442932

RESUMO

BACKGROUND: Loeys-Dietz syndrome (LDS) is a rare autosomal-dominant connective tissue disorder caused by genetic mutations in the transforming growth factor-ß (TGFß) signaling pathway. In addition to vascular malformations, patients with LDS commonly present with bone and tendon abnormalities, including joint laxity. While TGFß signaling dysregulation has been implicated in many of these clinical manifestations, the degree to which it influences the tendinopathy and tendon healing issues in LDS has not been determined. METHODS: Wound healing after patellar tendon transection was compared between wild-type (WT) and Tgfbr2-mutant (LDS) mice (7 mice per group). In all mice, the right patellar tendon was transected at midsubstance, while the left was untouched to serve as a control. Mice were euthanized 6 weeks after surgery. Tendon specimens were harvested for histopathologic grading according to a previously validated scoring metric, and gene expression levels of Mmp2, Tgfb2, and other TGFß-signaling genes were assayed. Between-group comparisons were made using 1-way analysis of variance with post hoc Tukey honestly significant difference testing. RESULTS: Expression levels of assayed genes were similar between LDS and WT tendons at baseline; however, at 6 weeks after patellar tendon transection, LDS tendons showed sustained elevations in Mmp2 and Tgfb2 compared with baseline values; these elevations were not seen in normal tendons undergoing the same treatments. Histologically, untreated LDS tendons had significantly greater cellularity and cell rounding compared with untreated WT tendons, and both WT and LDS tendons had significantly worse histologic scores after surgery. CONCLUSION: We present the first mechanistic insight into the effect of LDS on tendons and tendon healing. The morphologic differences between LDS and WT tendons at baseline may help explain the increased risk of tendon/ligament dysfunction in patients with LDS, and the differential healing response to injury in LDS may account for the delayed healing and weaker repair tissue. LEVEL OF EVIDENCE: Level V.


Assuntos
Síndrome de Loeys-Dietz , Ligamento Patelar , Fator de Crescimento Transformador beta2 , Animais , Modelos Animais de Doenças , Síndrome de Loeys-Dietz/genética , Metaloproteinase 2 da Matriz , Camundongos , Ligamento Patelar/fisiopatologia , Ligamento Patelar/cirurgia , Tendões/fisiopatologia , Tendões/cirurgia , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/fisiologia , Cicatrização
2.
J Biol Chem ; 297(3): 101070, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34389355

RESUMO

Transforming growth factor-beta 2 (TGF-ß2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-ß2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-ß2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-ß2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-ß2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm's canal (MSC) cell monolayers was decreased by TGF-ß2 treatment. SAHA inhibited the effects of TGF-ß2 on the permeability of these cells. TGF-ß2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-ß2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-ß2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-ß signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-ß. These results imply that SAHA prevents TGF-ß2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-ß signaling in TM and MSC cells.


Assuntos
Fator de Crescimento Transformador beta2/metabolismo , Vorinostat/metabolismo , Vorinostat/farmacologia , Animais , Humor Aquoso/metabolismo , Humor Aquoso/fisiologia , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Macaca fascicularis , Masculino , Hipertensão Ocular/metabolismo , Fosforilação , Cultura Primária de Células , Coelhos , Transdução de Sinais , Suínos , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/fisiologia , Fatores de Crescimento Transformadores/metabolismo
3.
J Biomed Sci ; 28(1): 47, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140021

RESUMO

BACKGROUND: Elevated transforming growth factor (TGF)-ß2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-ß2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators. METHODS: The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-ß levels were measured using the Bio-Plex Pro TGF-ß Assay. RNA scope was used to assess the expression of ATX and TGF-ß2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-ß2 regulates ATX expressions. RESULTS: TGF-ß2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-ß2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-ß2 with concentration-dependent manner. In counter, ATX also induced TGF-ß1, TGF-ß2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-ß and ATX inhibition. CONCLUSIONS: Trans-signaling of TGF-ß2 regulates ATX expressions and thereby induced upregulations of TGF-ßs or fibrosis of hTM. TGF-ß2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.


Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glaucoma de Ângulo Aberto/classificação , Humanos , Masculino , Pessoa de Meia-Idade
4.
J Invest Dermatol ; 141(11): 2679-2689, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34029574

RESUMO

Opsin-3 (OPN3) is a potential key regulator of human melanocyte melanogenesis. How OPN3-mediated regulation of melanocyte melanogenesis is triggered is largely unclear. TGFß can inhibit the growth of human melanocytes and reduce melanin synthesis in melanocytes. However, whether TGFß2 can modulate pigmentation in normal human primary melanocytes through OPN3 is entirely unknown. In this study, we constructed a coculture model with human epidermal melanocytes and keratinocytes. OPN3, tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2 expression and TYR activity were detected to be higher in cocultured cells than in monocultured cells. Moreover, elevated levels of TGFß2 were detected in the culture supernatant of melanocytes cocultured with keratinocytes. OPN3 inhibition in melanocytes decreased TYR, TRP-1, and TRP-2 expression and downregulated TYR activity. Our findings indicate that TGFß2 upregulates TYR activity and TRP-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. Interestingly, treatment with the TGFß2 receptor inhibitor LY2109761 (10 µM) did not inhibit TGFß2-induced melanocyte melanogenesis though OPN3. Collectively, our data suggest that TGFß2 upregulates TYR activity through OPN3 through a TGFß2 receptor-independent and calcium-dependent G-protein coupled signaling pathway.


Assuntos
Melanócitos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Opsinas de Bastonetes/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Humanos , Oxirredutases Intramoleculares/análise , Queratinócitos/metabolismo , Oxirredutases/análise , Transdução de Sinais/fisiologia , Regulação para Cima
5.
Exp Eye Res ; 207: 108594, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33894227

RESUMO

The TGF beta-1, -2 and -3 isoforms are transcribed from different genes but bind to the same receptors and signal through the same canonical and non-canonical signal transduction pathways. There are numerous regulatory mechanisms controlling the action of each isoform that include the organ-specific cells producing latent TGF beta growth factors, multiple effectors that activate the isoforms, ECM-associated SLRPs and basement membrane components that modulate the activity and localization of the isoforms, other interactive cytokine-growth factor receptor systems, such as PDGF and CTGF, TGF beta receptor expression on target cells, including myofibroblast precursors, receptor binding competition, positive and negative signal transduction effectors, and transcription and translational regulatory mechanisms. While there has long been the view that TGF beta-1and TGF beta-2 are pro-fibrotic, while TGF beta-3 is anti-fibrotic, this review suggests that view is too simplistic, at least in adult tissues, since TGF beta-3 shares far more similarities in its modulation of fibrotic gene expression with TGF beta-1 and TGF beta-2, than it does differences, and often the differences are subtle. Rather, TGF beta-3 should be seen as a fibro-modulatory partner to the other two isoforms that modulates a nuanced and better controlled response to injury. The complex interplay between the three isoforms and numerous interactive proteins, in the context of the cellular milieu, controls regenerative non-fibrotic vs. fibrotic healing in a response to injury in a particular organ, as well as the resolution of fibrosis, when that occurs.


Assuntos
Córnea/patologia , Fator de Crescimento Transformador beta1/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Fator de Crescimento Transformador beta3/fisiologia , Animais , Córnea/metabolismo , Fibrose/metabolismo , Humanos , Isoformas de Proteínas
6.
Nutrients ; 12(4)2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32326558

RESUMO

Human colostrum (HC) is a rich source of immune mediators that play a role in immune defences of a newly born infant. The mediators include transforming growth factor ß (TGF-ß) which exists in three isoforms that regulate cellular homeostasis and inflammation, can induce or suppress immune responses, limit T helper 1 cells (Th1) reactions and stimulate secretory immunoglobulin A (IgA) production. Human milk TGF-ß also decreases apoptosis of intestinal cells and suppresses macrophage cytokine expression. The aim of the study was to determine the concentration of TGF-ß2 in HC obtained from the mothers who delivered vaginally (VD) or by caesarean section (CS), and to compare the concentrations in HC from mothers who delivered at term (TB) or preterm (PB). In this study, 56% of preterm pregnancies were delivered via CS. The concentrations of TGF-ß2 were measured in HC from 299 women who delivered in the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw: 192 (VD), 107 (CS), 251 (TB), and 48 (PB). The colostrum samples were collected within 5 days post-partum. TGF-ß2 levels in HC were measured by the enzyme-linked immunosorbent assay (ELISA) test with the Quantikine ELISA Kit-Human TGF-ß2 (cat.no. SB250). Statistical significance between groups was calculated by the Student t-test using StatSoft Statistica 13 software. The mean TGF-ß2 concentration in patients who delivered at term or preterm were comparable. The levels of TGF-ß2 in HC were higher after preterm than term being 4648 vs. 3899 ng/mL (p = 0.1244). The delivery via CS was associated with higher HC concentrations of TGF-ß2. The levels of TGF-ß2 were significantly higher in HC after CS than VD (7429 vs. 5240 ng/mL; p = 0.0017). The data from this study suggest: caesarean section was associated with increased levels of TGF-ß2 in HC. The increased levels of TGF-ß2 in HC of women who delivered prematurely require further research. Early and exclusive breast-feeding by mothers after caesarean section and premature births with colostrum containing high TGF-ß2 levels may prevent the negative impact of pathogens which often colonize the gastrointestinal tract and may reduce the risk of chronic diseases in this group of patients.


Assuntos
Cesárea , Colostro/química , Trabalho de Parto Prematuro/metabolismo , Período Pós-Parto/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Aleitamento Materno , Doença Crônica , Colostro/imunologia , Feminino , Gastroenterite/microbiologia , Gastroenterite/prevenção & controle , Humanos , Recém-Nascido , Gravidez , Nascimento Prematuro/imunologia , Estudos Prospectivos , Risco , Fator de Crescimento Transformador beta2/imunologia , Fator de Crescimento Transformador beta2/fisiologia
7.
Biochem Biophys Res Commun ; 508(3): 889-893, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30538046

RESUMO

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)ß2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFß2 for 21 days. At 3 days, TGFß2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFß2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs.


Assuntos
Caderinas/metabolismo , Conexina 43/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Fibroblastos/ultraestrutura , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Camundongos
8.
Arterioscler Thromb Vasc Biol ; 39(2): 212-223, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30580570

RESUMO

Objective- Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40-/- (interleukin 12 p40) mice, we investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm. Approach and Results- Male (8-10 week-old) wild-type and IL12p40-/- mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40-/- mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40-/- mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented the Tgfß2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40-/- mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions- IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfß2.


Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Subunidade p40 da Interleucina-12/fisiologia , Animais , Colágeno/metabolismo , Subunidade p40 da Interleucina-12/deficiência , Macrófagos/fisiologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta2/fisiologia , Rigidez Vascular
9.
Development ; 144(23): 4377-4385, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038307

RESUMO

Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms.


Assuntos
Rim/embriologia , Morfogênese/fisiologia , Ureter/embriologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 7/deficiência , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/fisiologia , Imageamento Tridimensional , Conceitos Matemáticos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Modelos Biológicos , Morfogênese/genética , Mutação , Fenótipo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Fator de Crescimento Transformador beta2/deficiência , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/fisiologia
10.
BMC Cancer ; 17(1): 650, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28915803

RESUMO

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness. METHODS: MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes. RESULTS: We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFß) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFß isoform 2 (TGFß2), TGFß receptor type 1 (TßR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical inhibition demonstrated that TRAP-dependent migration and proliferation is regulated via TGFß2/TßR, whereas proliferation beyond basal levels is regulated through CD44. CONCLUSION: Altogether, TRAP promotes metastasis-related cell properties in MDA-MB-231 breast cancer cells via TGFß2/TßR and CD44, thereby identifying a potential signaling mechanism associated to TRAP action in breast cancer cells.


Assuntos
Receptores de Hialuronatos/metabolismo , Fosfatase Ácida Resistente a Tartarato/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Forma Celular , Feminino , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Transdução de Sinais
11.
Cornea ; 36 Suppl 1: S41-S45, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28902018

RESUMO

Because human corneal endothelial cells (HCECs) do not proliferate once the endothelial monolayer has formed, corneal wound healing is believed to be mediated by cell enlargement or migration, rather than by proliferation. However, the cellular mechanisms involved in wound healing by HCECs have not been fully determined. In this review, we focus on the effects of promyelocytic leukemia zinc finger (PLZF), a DNA-binding transcription factor, and transforming growth factor (TGF)-ß2 on the proliferation and migration of cultured HCECs. Involvement of the mitogen-activated protein kinase (MAPK) signaling pathway in the migration of HCECs was also investigated. Expression of PLZF mRNA decreased as cell-cell contact was disrupted and returned to the original level as cell-cell contact was re-formed. Assessment with a real-time cell electronic sensing system revealed that proliferation of cultured HCECs was inhibited after infection with Ad-PLZF and exposure to TGF-ß2. Migration of cultured HCECs was increased by TGF-ß2 through p38 MAPK activation. We conclude that PLZF expression in cultured HCECs is closely related to the formation of cell-cell contact and that TGF-ß2 suppresses proliferation of cultured HCECs, while promoting their migration through p38 MAPK activation.


Assuntos
Proliferação de Células/fisiologia , Endotélio Corneano/citologia , Movimento Celular/fisiologia , Células Cultivadas , Endotélio Corneano/metabolismo , Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Humanos , Proteína com Dedos de Zinco da Leucemia Promielocítica/fisiologia , RNA Mensageiro/genética , Fator de Crescimento Transformador beta2/fisiologia , Cicatrização/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
J Clin Invest ; 127(11): 3937-3953, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28945200

RESUMO

In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF-producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2-/- NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-ß2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-ß2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-ß2.


Assuntos
Monócitos/fisiologia , Esclerose Múltipla/metabolismo , Células-Tronco Neurais/transplante , Fator de Crescimento Transformador beta2/fisiologia , Animais , Encéfalo/imunologia , Encéfalo/patologia , Diferenciação Celular , Células Cultivadas , Citocinas/biossíntese , Citocinas/metabolismo , Células Dendríticas/fisiologia , Feminino , Imunomodulação , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microglia/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Células-Tronco Neurais/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Transcriptoma
13.
Exp Eye Res ; 164: 55-63, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28789942

RESUMO

Statins are cholesterol lowering drugs and have shown beneficial effects on glaucoma. With regard to the mechanism of statin action on glaucoma, we investigated the effects of statins on transforming growth factor-beta 2 (TGF-ß2)-induced expression of extracellular matrix (ECM) proteins in human astrocytes of the optic nerve head (ONH) lamina cribrosa (LC). By using primary human ONH astrocytes, we found that both simvastatin and lovastatin inhibited TGF-ß2-mediated expression of ECM proteins such as connective tissue growth factor, collagen I, fibronectin, and plasminogen activator inhibitor-1. Suppression of ECM related proteins is due to inhibition of Smad2/3 activation as statins inhibit TGF-ß2-induced Smad2 phosphorylation and Smad2/3 nuclear accumulation. In ONH astrocytes, TGF-ß2 does not induce MAPK activation. In this study we found an anti-fibrotic effect of statins in human astrocytes of the ONH and identified TGF-ß2 as a mediator of statin action, which may support a beneficial role for statins in blocking glaucomatous axonal damage induced by ECM remodeling.


Assuntos
Astrócitos/efeitos dos fármacos , Matriz Extracelular/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Disco Óptico/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Análise de Variância , Astrócitos/metabolismo , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Proteínas do Olho/metabolismo , Humanos , Lovastatina , Disco Óptico/citologia , Sinvastatina , Fator de Crescimento Transformador beta2/metabolismo
14.
Bull Exp Biol Med ; 162(5): 671-675, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28361415

RESUMO

The study established enhanced expression of vascular endothelial growth factor (VEGF) in the subpopulation of osteoblasts located in the regeneration region of femoral bone fracture near the titanium implants with bioactive calcium phosphate and hydroxyapatite coatings and suppressed activity of transforming growth factor-ß2 (TGF-ß2) in chondroblasts during the two weeks after surgery. In the delayed posttraumatic period, the distribution of TGF-ß2 inversely related to its maximal activity. The data revealed the up-regulating effect of bioresorbable coatings on expression of VEGF and TGF-ß2 and their implication in the control over various stages of reparative osteogenesis.


Assuntos
Regeneração Óssea , Parafusos Ósseos , Osso e Ossos/metabolismo , Materiais Revestidos Biocompatíveis/química , Fator de Crescimento Transformador beta2/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Fosfatos de Cálcio/química , Durapatita/química , Consolidação da Fratura , Masculino , Ratos , Titânio
15.
Invest Ophthalmol Vis Sci ; 58(2): 1288-1295, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28241317

RESUMO

Purpose: Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor ß2 (TGFß2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFß2 signaling pathways in CLAN formation. Methods: Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFß2, with or without the inhibitors of TGFß receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFß2 plus inhibitors for 10 days or pretreated with TGFß2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4',6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results: TGFß2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFß receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFß receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions: TGFß2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.


Assuntos
Actinas/metabolismo , Proteína Smad3/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Análise de Variância , Humor Aquoso/metabolismo , Western Blotting , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta , Transdução de Sinais/fisiologia , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/fisiologia
16.
Invest Ophthalmol Vis Sci ; 57(8): 3665-73, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27403995

RESUMO

PURPOSE: Transforming growth factor-ß induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-ß-driven EMT is dependent on the expression of the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-ß-induced lens EMT and determine whether it is required for this pathologic process. METHODS: Rat lens epithelial explants were used to investigate the role of Nox4 in TGF-ß-driven lens EMT. Nox1-4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH-oxidase-produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-ß-driven EMT. RESULTS: We demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-ß treatment induces Nox4 (but not Nox1-3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-ß treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-ß treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression. CONCLUSIONS: Transforming growth factor-ß drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Cristalino/fisiologia , NADPH Oxidases/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Células Epiteliais/fisiologia , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta2/fisiologia
17.
Dev Biol ; 415(1): 14-23, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27180663

RESUMO

The secondary palate separates the oral from the nasal cavity and its closure during embryonic development is sensitive to genetic perturbations. Mice with deleted Foxf2, encoding a forkhead transcription factor, are born with cleft palate, and an abnormal tongue morphology has been proposed as the underlying cause. Here, we show that Foxf2(-/-) maxillary explants cultured in vitro, in the absence of tongue and mandible, failed to close the secondary palate. Proliferation and collagen content were decreased in Foxf2(-/-) palatal shelf mesenchyme. Phosphorylation of Smad2/3 was reduced in mutant palatal shelf, diagnostic of attenuated canonical Tgfß signaling, whereas phosphorylation of p38 was increased. The amount of Tgfß2 protein was diminished, whereas the Tgfb2 mRNA level was unaltered. Expression of several genes encoding extracellular proteins important for Tgfß signaling were reduced in Foxf2(-)(/)(-) palatal shelves: a fibronectin splice-isoform essential for formation of extracellular Tgfß latency complexes; Tgfbr3 - or betaglycan - which acts as a co-receptor and an extracellular reservoir of Tgfß; and integrins αV and ß1, which are both Tgfß targets and required for activation of latent Tgfß. Decreased proliferation and reduced extracellular matrix content are consistent with diminished Tgfß signaling. We therefore propose that gene expression changes in palatal shelf mesenchyme that lead to reduced Tgfß signaling contribute to cleft palate in Foxf2(-)(/)(-) mice.


Assuntos
Fissura Palatina/embriologia , Fatores de Transcrição Forkhead/fisiologia , Mesoderma/embriologia , Palato/embriologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Animais , Colágeno/fisiologia , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fibronectinas/fisiologia , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrinas/fisiologia , Mandíbula/embriologia , Maxila/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoglicanas/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Língua/anormalidades , Língua/embriologia , Fator de Crescimento Transformador beta2/biossíntese , Fator de Crescimento Transformador beta2/genética
18.
Exp Eye Res ; 148: 97-102, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27091054

RESUMO

Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFß signaling pathway activator TGFß2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFß/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFß/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 µg/ml TGFß2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFß2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFß2-induced TGFß/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or ß-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFß2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFß/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and ß-catenin.


Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta2/fisiologia , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/fisiologia , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Humanos , Luciferases/metabolismo , Masculino , Transdução de Sinais/fisiologia , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Proteína Wnt3A/metabolismo , Proteína Wnt3A/farmacologia , beta Catenina/metabolismo
19.
Neuron ; 86(5): 1228-39, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-26050041

RESUMO

Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-ßr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-ßr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-ßr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network.


Assuntos
Aplysia/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Espaço Intracelular/fisiologia , Memória de Longo Prazo/fisiologia , Animais , Glicoproteínas de Membrana/fisiologia , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptor trkB , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/fisiologia , Fatores de Tempo , Fator de Crescimento Transformador beta2/fisiologia
20.
Reprod Toxicol ; 50: 129-33, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25450421

RESUMO

Cleft palate is a common birth defect affecting 1 in 700 births. Transforming growth factor-ßs (TGF-ßs) are important signaling molecules, and their functions in murine palate development have received great attention. TGF-ß3 is expressed exclusively in palatal epithelial cells and mediates epithelial fusion, whereas the importance of TGF-ß1 and 2 in palate have not yet been demonstrated in vivo, since inactivation of Tgf-ß1 or Tgf-ß2 genes in mice did not reveal significant palate defects. We hypothesized that TGF-ß1 and TGF-ß2 can compensate each other during palate formation. To test this, we generated Tgf-ß1 and Tgf-ß2 compound mutant mice and found that approximately 40% of [Tgf-ß1(+/-); Tgf-ß2(-/-)] compound mutant embryos display cleft palate on C57 background. In addition, 26% of Tgf-ß2(-/-) embryos on 129 background, but not in C57 or Black Swiss, displayed cleft palate. TGF-ß1 and 2 functions are required for murine palate development in strain-dependent manner.


Assuntos
Palato/embriologia , Fator de Crescimento Transformador beta1/fisiologia , Fator de Crescimento Transformador beta2/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie
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