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1.
J Cell Sci ; 130(20): 3481-3495, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28851806

RESUMO

Scarring and fibrotic disease result from the persistence of myofibroblasts characterized by high surface expression of αv integrins and subsequent activation of the transforming growth factor ß (TGFß) proteins; however, the mechanism controlling their surface abundance is unknown. Genetic screening revealed that human primary stromal corneal myofibroblasts overexpress a subset of deubiquitylating enzymes (DUBs), which remove ubiquitin from proteins, preventing degradation. Silencing of the DUB USP10 induces a buildup of ubiquitin on integrins ß1 and ß5 in cell lysates, whereas recombinant USP10 removes ubiquitin from these integrin subunits. Correspondingly, the loss and gain of USP10 decreases and increases, respectively, αv/ß1/ß5 protein levels, without altering gene expression. Consequently, endogenous TGFß is activated and the fibrotic markers alpha-smooth muscle actin (α-SMA) and cellular fibronectin (FN-EDA) are induced. Blocking either TGFß signaling or cell-surface αv integrins after USP10 overexpression prevents or reduces fibrotic marker expression. Finally, silencing of USP10 in an ex vivo cornea organ culture model prevents the induction of fibrotic markers and promotes regenerative healing. This novel mechanism puts DUB expression at the head of a cascade regulating integrin abundance and suggests USP10 as a novel antifibrotic target.


Assuntos
Cadeias beta de Integrinas/metabolismo , Integrina beta1/metabolismo , Ubiquitina Tiolesterase/fisiologia , Ubiquitinação , Animais , Células Cultivadas , Células HEK293 , Humanos , Proteólise , Transdução de Sinais , Sus scrofa , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta/fisiologia , Cicatrização
2.
Am J Hum Genet ; 92(6): 1001-7, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23731542

RESUMO

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.


Assuntos
Genes Dominantes , Mutação de Sentido Incorreto , Miofibromatose/congênito , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Estudos de Associação Genética , Humanos , Masculino , Miofibromatose/genética , Linhagem , Receptor Notch3 , Receptores Notch/genética , Análise de Sequência de DNA
3.
Cell Signal ; 113: 110963, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37931692

RESUMO

Following wounding, endogenously secreted TGFßs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFß effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFß. TGFß also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFß1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFß1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFß1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFß1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10-15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFß pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.


Assuntos
Zíper de Leucina , Miofibroblastos , Humanos , Fosforilação , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Smad2/metabolismo
4.
Eur J Cell Biol ; 102(4): 151347, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37562219

RESUMO

The differentiation of fibroblasts into pathological myofibroblasts during wound healing is characterized by increased cell surface expression of αv-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from αv-integrins, leading to cell surface integrin accumulation, subsequent TGFß1 activation, and pathological myofibroblast differentiation. In this study, a yeast two-hybrid screen revealed a novel binding partner for USP10, the formin, DAAM1. We found that DAAM1 binds to and inhibits USP10's DUB activity through the FH2 domain of DAAM1 independent of its actin functions. The USP10/DAAM1 interaction was also supported by proximity ligation assay (PLA) in primary human corneal fibroblasts. Treatment with TGFß1 significantly increased USP10 and DAAM1 protein expression, PLA signal, and co-localization to actin stress fibers. DAAM1 siRNA knockdown significantly reduced co-precipitation of USP10 and DAAM1 on purified actin stress fibers, and ß1- and ß5-integrin ubiquitination. This resulted in increased αv-, ß1-, and ß5-integrin total protein levels, αv-integrin recycling, and extracellular fibronectin (FN) deposition. Together, our data demonstrate that DAAM1 inhibits USP10's DUB activity on integrins subsequently regulating cell surface αv-integrin localization and FN accumulation.


Assuntos
Integrinas , Humanos , Actinas/metabolismo , Enzimas Desubiquitinantes/metabolismo , Forminas/metabolismo , Integrinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Cicatrização
5.
Curr Eye Res ; 47(8): 1165-1178, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35481448

RESUMO

PURPOSE: Transforming growth factor-beta 2 (TGFß2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma (POAG). TGFß2 activates extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate HTM cell contractility is unclear. Here, we investigated the molecular mechanism underlying TGFß2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways with different culture substrates. METHODS: Hydrogels were engineered by mixing collagen type I, elastin-like polypeptide, and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Primary HTM cells were seeded atop pre-formed hydrogels for comparisons with glass, or encapsulated within the hydrogels. Changes in actin cytoskeleton, extracellular matrix (ECM) production, phospho-myosin light chain (p-MLC) levels, and hydrogel contraction were assessed. RESULTS: HTM cell morphology and filamentous (F)-actin organization were affected by the underlying culture substrates. TGFß2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, α-smooth muscle actin (αSMA), fibronectin (FN), and p-MLC in HTM cells. ERK inhibition, even as short as 4 h, further increased TGFß2-induced p-MLC in HTM cells on hydrogels, but not on glass. This translated into hypercontractility of HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGFß2-induced hydrogels. CONCLUSIONS: Our data suggest that ERK signaling negatively regulates ROCK-mediated HTM cell contractility. These findings emphasize the critical importance of using tissue-mimetic ECM substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro.


Assuntos
Glaucoma de Ângulo Aberto , Malha Trabecular , Actinas/metabolismo , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Hidrogéis/farmacologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta2/farmacologia
6.
Mol Vis ; 17: 2835-46, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22128231

RESUMO

PURPOSE: There is an unmet challenge to promote wound healing in non-healing wounds such as in the post-LASIK (laser-assisted in situ keratomileusis) cornea. Using human corneal fibroblasts (HCFs) in cell culture, we investigated the concentration dependence of the growth factor transforming growth factor ß1 (TGFß1) on wound closure. Although high concentrations of TGFß1 leads to scarring, we asked whether low concentrations of TGFß1 could promote wound healing without generating a large fibrotic response. METHODS: HCFs were cultured in supplemented serum-free media (SSFM). Cell migration was assessed by scratch-wounding. SMAD 2/3 and p38 mitogen-activated protein kinase (p38MAPK) localization and α-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. Active TGFß was quantified using a luciferase bio-assay. RESULTS: We found that neutralizing antibody to TGFß1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGFß1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGFß1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGFß1 (0.01-1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGFß1) significantly decreased cell migration (63%-86%), induced myofibroblast differentiation (83%-88%), increased SMAD 2/3 localization into the nucleus (72%-79%) and inhibited the activation of p38MAPK (51%-63%). In contrast, addition of the lower concentration of TGFß1 (0.01 ng/ml TGFß1) promoted a cell migration rate that was similar to endogenous TGFß, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGFß1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGFß1-induced cell migration. CONCLUSIONS: Together, our data demonstrate that low concentrations of TGFß1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGFß may be useful in treating non-healing corneal wounds.


Assuntos
Movimento Celular/efeitos dos fármacos , Córnea/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/genética , Actinas/metabolismo , Anticorpos Bloqueadores/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Córnea/citologia , Córnea/metabolismo , Córnea/cirurgia , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Ativação Enzimática , Fibroblastos/citologia , Humanos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/fisiologia , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética
7.
Exp Cell Res ; 316(14): 2322-39, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20580707

RESUMO

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.


Assuntos
Córnea/citologia , Córnea/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Sindecana-1/genética , Sindecana-1/metabolismo , Animais , Adesão Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Cadeias alfa de Integrinas/metabolismo , Integrina beta1/metabolismo , Cloreto de Magnésio/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Reação em Cadeia da Polimerase
8.
Invest Ophthalmol Vis Sci ; 62(13): 15, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34665194

RESUMO

Purpose: Integrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFß activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs). Methods: Primary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry. Results: USP10 overexpression increased α5ß1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7-2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5ß1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN. Conclusions: Our data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.


Assuntos
Córnea/metabolismo , Fibronectinas/metabolismo , Ubiquitina Tiolesterase/biossíntese , Adesão Celular , Membrana Celular/metabolismo , Células Cultivadas , Córnea/citologia , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Transdução de Sinais
9.
J Biol Chem ; 284(31): 20708-17, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19487690

RESUMO

Fibrosis is characterized by elevated transforming growth factor beta (TGFbeta) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin alphavbeta3 from the cell surface. Since increased alphavbeta3 expression correlates with increased TGFbeta signaling, we hypothesized that aberrant PAI-1-mediated alphavbeta3 endocytosis could initiate an autocrine loop of TGFbeta activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), alphavbeta3 endocytosis was reduced by approximately 75%, leaving alphavbeta3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFbeta signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFbeta activity that correlated with an increase in alphavbeta3 and TGFbeta receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFbeta activation, as evidenced by a mere 25% reduction in TGFbeta activity when plasmin was inhibited. Treatment of cells with an alphavbeta3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFbeta inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFbeta by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and beta3 overexpression led to an increase in TGFbeta activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of alphavbeta3 integrin is critical for the control of TGFbeta signaling and the prevention of fibrotic disease.


Assuntos
Comunicação Autócrina , Integrina alfaVbeta3/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colágeno/biossíntese , DNA Complementar/metabolismo , Endocitose , Fibrinolisina/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Humanos , Camundongos , Camundongos Knockout , Vison , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
10.
Mol Biol Cell ; 18(7): 2716-27, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17507651

RESUMO

Fibroblasts migrate into and repopulate connective tissue wounds. At the wound edge, fibroblasts differentiate into myofibroblasts, and they promote wound closure. Regulated fibroblast-to-myofibroblast differentiation is critical for regenerative healing. Previous studies have focused on the role in fibroblasts of urokinase plasmingen activator/urokinase plasmingen activator receptor (uPA/uPAR), an extracellular protease system that promotes matrix remodeling, growth factor activation, and cell migration. Whereas fibroblasts have substantial uPA activity and uPAR expression, we discovered that cultured myofibroblasts eventually lost cell surface uPA/uPAR. This led us to investigate the relevance of uPA/uPAR activity to myofibroblast differentiation. We found that fibroblasts expressed increased amounts of full-length cell surface uPAR (D1D2D3) compared with myofibroblasts, which had reduced expression of D1D2D3 but increased expression of the truncated form of uPAR (D2D3) on their cell surface. Retaining full-length uPAR was found to be essential for regulating myofibroblast differentiation, because 1) protease inhibitors that prevented uPAR cleavage also prevented myofibroblast differentiation, and 2) overexpression of cDNA for a noncleavable form of uPAR inhibited myofibroblast differentiation. These data support a novel hypothesis that maintaining full-length uPAR on the cell surface regulates the fibroblast to myofibroblast transition and that down-regulation of uPAR is necessary for myofibroblast differentiation.


Assuntos
Diferenciação Celular , Fibroblastos/citologia , Mioblastos/citologia , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Actinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Córnea/citologia , Córnea/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Mioblastos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Fibras de Estresse/efeitos dos fármacos , Sulfonas/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
11.
Mol Ther Nucleic Acids ; 21: 1029-1043, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32829179

RESUMO

Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (self-deliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. αv integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor ß (TGF-ß) activation. Previously, we demonstrated that αv integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45+ cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.

12.
Adv Protein Chem Struct Biol ; 118: 273-288, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31928728

RESUMO

Exfoliation syndrome (XFS) is an age-related disease defined by the deposition of aggregated fibrous material (XFM) in the peri-cellular space. Principal morbidity occurs in the eye, where XFM accumulates on the anterior ocular tissues. GWAS have found that certain genetic variants of lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme that is required for elastic fiber formation confer risk for the development of XFS, but are not a single causative factor as many genetically affected individuals do not develop XFS or subsequent glaucoma (XFG). We have found that XFG cells display defects in lysosomes, microtubules, autophagy, and mitochondria resembling defects found in cells from age-related syndromes, such as the main neurodegenerative diseases. In the majority of these diseases, the determining cellular factor is a protein containing intrinsically disordered regions (IDRs) and displaying a high propensity for aggregation. We have found that in XFG patient-derived cells, LOXL1 protein is actively subjected to autophagic clearance, suggesting that LOXL1 is undergoing aggregation. In silico analysis demonstrates that LOXL1's first 369 aa constitute an IDR with the highest disorder probability peak centering around the known risk positions. Experimentally, we have found over-expression of either unmodified LOXL1 or fluorescent chimeras preserving the well-structured N-terminus cause copious intracellular aggregation and that aggregation wanes when the high IDR peaks are deleted. Overall, our work suggests that XFS/G results from the aggregation of the LOXL1 protein coupled with a reduction of cellular proteostasis capabilities in aging, resulting in a chronic build-up of LOXL1-containing protein aggregates.


Assuntos
Aminoácido Oxirredutases/metabolismo , Síndrome de Exfoliação/metabolismo , Dobramento de Proteína , Autofagia , Síndrome de Exfoliação/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutação , Ligação Proteica
13.
J Vis Exp ; (144)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30829330

RESUMO

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.


Assuntos
Córnea/fisiopatologia , Técnicas de Cultura de Órgãos/métodos , Animais , Modelos Animais de Doenças , Suínos , Transfecção
14.
J Glaucoma ; 27(3): 197-201, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-27977481

RESUMO

In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.


Assuntos
Autofagia/fisiologia , Síndrome de Exfoliação/etiologia , Glaucoma/etiologia , Síndrome de Exfoliação/metabolismo , Síndrome de Exfoliação/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/fisiologia , Glaucoma/metabolismo , Glaucoma/patologia , Humanos , Pressão Intraocular/fisiologia , Masculino , Cápsula de Tenon/metabolismo , Cápsula de Tenon/patologia
15.
J Glaucoma ; 27 Suppl 1: S44-S53, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29547474

RESUMO

Exfoliation syndrome (XFS) is an age-related disease involving the deposition of aggregated fibrillar material (exfoliation material) at extracellular matrices in tissues that synthesize elastic fibers. Its main morbidity is in the eye, where exfoliation material accumulations form on the surface of the ciliary body, iris, and lens. Exfoliation glaucoma (XFG) occurs in a high proportion of persons with XFS and can be a rapidly progressing disease. Worldwide, XFG accounts for about 25% of open-angle glaucoma cases. XFS and XFG show a sharp age-dependence, similarly to the many age-related diseases classified as aggregopathies. Progress in understanding the cellular bases for XFS/XFG has been slowed by a lack of experimental models. Working with primary human tenon fibroblasts (TF) derived from trabeculectomies of XFG patients and age-matched primary open-glaucoma controls, we found that TF from XFG cells display many of the functional features observed in cells from other protein aggregate diseases, such as Parkinson, Alzheimer, Huntington, and age-related macular degeneration. We have documented defects in lysosomal positioning, microtubule organization, autophagy processing rate, and mitochondrial health. In regard to failure of lysosomal and autophagosome positioning in XFG cells, we have found that XFG TF are unable to establish the transnuclear microtubule organizing center that is required for efficient centripetal vesicular locomotion along microtubules. In regard to potential sources of the autophagy malfunction, we have directed our attention to a potential role of the lysyl oxidase-like 1 protein (LOXL1), the elastic fiber catalyst that displays variant-dependent association with risk for XFG. Our experiments show that (a) in XFG cells, a substantial fraction of LOXL1 is processed for degradation by the autophagic system; (b) most of the LOXL1 N-terminus domain exists in a highly disordered state, a condition known to greatly increase the frequency of polypeptide misfolding; (c) that maximum misfolding occurs at amino acid position 153, the location of the high risk variant G153D; and (d) that replacement of glycine (G) by aspartate (D) there results in a substantial decrease in disorder within the 20 amino acid surrounding domain. Finally, we show that clusterin, a protein that can be induced by the presence of intracellular, or extracellular aggregates, is uniformly overexpressed in XFG TF. The implications of our results for a theory relating XFG to cellular aggregopathy are discussed.


Assuntos
Aminoácido Oxirredutases/metabolismo , Autofagia , Síndrome de Exfoliação/enzimologia , Glaucoma de Ângulo Aberto/enzimologia , Segmento Anterior do Olho/patologia , Síndrome de Exfoliação/patologia , Matriz Extracelular/patologia , Glaucoma de Ângulo Aberto/patologia , Humanos , Agregados Proteicos
16.
FASEB J ; 20(7): 1006-8, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16585062

RESUMO

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFbeta) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGFbeta induces expression of smooth muscle alpha actin (SMalphaA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFbeta induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGFbeta-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFbeta-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK -/- MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMalphaA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK -/- [corrected]MEFs. We conclude that FAK does not contribute to TGFbeta-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SMalphaA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.


Assuntos
Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Actinas/metabolismo , Diferenciação Celular , Células Cultivadas , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/genética , Deleção de Genes , Regulação da Expressão Gênica , Interferência de RNA , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
17.
Stem Cells Int ; 2016: 5309484, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26839562

RESUMO

Stem cells based tissue engineering requires biocompatible materials, which allow the cells to adhere, expand, and differentiate in a large scale. An ideal biomaterial for clinical application should be free from mammalian products which cause immune reactivities and pathogen infections. We invented a novel biodegradable poly(L-lactic-co-ε-caprolactone)-sericin (PLCL-SC) copolymer membrane which was fabricated by electrospinning. Membranes with concentrations of 2.5 or 5% (w/v) SC exhibited qualified texture characteristics with a noncytotoxic release profile. The hydrophilic properties of the membranes were 35-40% higher than those of a standard PLCL and commercial polystyrene (PS). The improved characteristics of the membranes were due to an addition of new functional amide groups, C=O, N-H, and C-N, onto their surfaces. Degradation of the membranes was controllable, depending on the content proportion of SC. Results of thermogram indicated the superior stability and crystallinity of the membranes. These membranes enhanced human Wharton's jelly mesenchymal stem cells (hWJMSC) proliferation by increasing cyclin A and also promoted cell adhesion by upregulating focal adhesion kinase (FAK). On the membranes, hWJMSC differentiated into a neuronal lineage with the occurrence of nestin. These data suggest that PLCL-SC electrospun membrane represents some properties which will be useful for tissue engineering and medical applications.

18.
PLoS One ; 11(7): e0157404, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27391778

RESUMO

PURPOSE: To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma. METHODS: Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal. RESULTS: In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8-10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3-8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02). CONCLUSIONS: Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.


Assuntos
Autofagia , Síndrome de Exfoliação/cirurgia , Fibroblastos/metabolismo , Glaucoma de Ângulo Aberto/cirurgia , Idoso , Idoso de 80 Anos ou mais , Aminoácido Oxirredutases/genética , Pré-Escolar , Síndrome de Exfoliação/metabolismo , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Pressão Intraocular , Luz , Masculino , Potenciais da Membrana , Pessoa de Meia-Idade , Membranas Mitocondriais/metabolismo , Espalhamento de Radiação , Estrabismo/cirurgia
19.
Invest Ophthalmol Vis Sci ; 46(1): 96-103, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15623760

RESUMO

PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.


Assuntos
Córnea/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Pseudópodes/metabolismo , Cicatrização , Animais , Western Blotting , Caderinas/metabolismo , Técnicas de Cultura de Células , Conexina 43/metabolismo , Lesões da Córnea , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Imunoprecipitação , Junções Intercelulares , Fosforilação , Coelhos , Proteína da Zônula de Oclusão-1
20.
Mol Vis ; 11: 859-68, 2005 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16270025

RESUMO

PURPOSE: Maintenance of avascularity of the normal cornea and control of neovascularization during wound healing depend on a balance of angiogenic and antiangiogenic factors. The purpose of this paper is to determine the ability of corneal cells to convert plasminogen to angiostatins and to compare these products with those made by intact corneas. METHODS: RT-PCR was performed using plasminogen specific primers and the generated cDNA was sequenced. The proteins in corneal extracts, cornea conditioned medium, and medium from corneal epithelial cells, stromal fibroblasts, and myofibroblasts incubated with plasminogen were separated by SDS-PAGE and electroblotted. Western blots used monoclonal antibodies to kringles 1-3 to detect plasminogen and angiostatins. Angiostatins were isolated and tested for activity in a vascular endothelial cell proliferation inhibition assay. RESULTS: Plasminogen, its mRNA and angiostatins were found in human corneal tissue extracts from the epithelial, stromal, and endothelial layers and from cornea conditioned medium, but not in medium from cultured epithelial cells, stromal fibroblasts, or myofibroblasts. However, cultures of corneal epithelial cells and stromal fibroblasts were able to convert exogenously added plasminogen to angiostatins, whereas cultured myofibroblasts did not. Angiostatins of 38 and 34 kDa were found under all angiostatin generating conditions; however other angiostatins differed in size. Further, the angiostatins isolated from fibroblast culture supernatants inhibited vascular endothelial cell proliferation. CONCLUSIONS: Conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a means of treating abnormal corneal neovascularization without the risk of side effects.


Assuntos
Angiostatinas/biossíntese , Substância Própria/metabolismo , Epitélio Corneano/metabolismo , Plasminogênio/metabolismo , Angiostatinas/genética , Angiostatinas/farmacologia , Western Blotting , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Substância Própria/citologia , Substância Própria/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas de Cultura de Órgãos , Plasminogênio/genética , Plasminogênio/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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