RESUMO
PURPOSE: Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic ß cells. In this study, we investigate the underlying mechanism. RESULTS: Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V-induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP. CONCLUSION: Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V-induced insulin biosynthesis and secretion in INS-1 cells.
Assuntos
Insulina , Ilhotas Pancreáticas , Receptor IGF Tipo 1 , Transdução de Sinais , Proteínas de Sinalização YAP , Animais , Colágeno Tipo V/farmacologia , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Receptor IGF Tipo 1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismoRESUMO
Extracellular matrix (ECM) molecules have multiple functions; prevention of cytotoxicity, provision of mechanical support, cell adhesive substrates and structural integrity in addition to mediation of cellular signaling. In this study, we report that the proliferation of INS-1 cells cultured on collagen I-coated dishes is enhanced, but it is inhibited on collagen V-coated dishes. Inhibitory proliferation on collagen V-coated is not due to apoptosis induction. Silibinin decreases hepatic glucose production and protects pancreatic ß-cells, as a potential medicine for type II diabetes. Silibinin up-regulates the proliferation of cells cultured on both collagen I- and V-coated dishes. Collagen-coating regulates gene expression of collagen in a collagen type-related manner. Silibinin increases mRNA expression of collagen I in the cells on collagen I- and V-coated dishes; however, silibinin decreases collagen V mRNA expression on collagen I- and V-coated dishes. Collagen I-coating significantly enhances nuclear translocation of ß-catenin, while collagen V-coating reduces it. Differential effects of silibinin on collagen I mRNA and collagen V mRNA can be accounted for by the finding that silibinin enhances nuclear translocation of ß-catenin on both collagen I- and V-coated dishes, since phenomenologically nuclear translocation of ß-catenin enhances collagen I mRNA but represses collagen V mRNA. These results demonstrate that nuclear translocation of ß-catenin up-regulates proliferation and collagen I gene expression, whereas it down-regulates collagen V gene expression of INS-1 cells. Differential gene expressions of collagen I and V by nuclear ß-catenin could be important for understanding fibrosis where collagen I and V may have differential effects.
Assuntos
Núcleo Celular/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo V/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Silibina/farmacologia , beta Catenina/metabolismo , Animais , Bovinos , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Because collagen type V (Col V) can be exposed in tissue injury, we hypothesized that oral administration of this collagen species modulates the inflammation and remodeling of experimental synovitis, avoiding joint destruction, and that the modulation may differ according to the temporal administration. Arthritis (IA, n = 20) was induced in Lewis rats by intraarticular (ia) injection of 500 µg of methylated bovine serum albumin (mBSA) emulsified in complete Freund's adjuvant (CFA) (10 µl) followed by an intraarticular booster of mBSA (50 µg) in saline (50 µl) administered at 7 and 14 days. The control group received saline (50 µl, ia). After the first intraarticular injection, ten IA animals were supplemented via gavage with Col V (500 µg/300 µl) daily for 30 days (IA/Suppl). The control group received saline (50 µL) and Col V supplement in the same way (Suppl). Col V oral administration in IA/Suppl led to 1) inhibited edema and severe inflammatory cell infiltration, 2) decreased collagen fiber content, 3) decreased collagen type I, 4) inhibited lymphocyte subpopulations and macrophages, 5) inhibited IL-1ß, IL-10, IL-17 and TNF-α production and 6) increased expression of caspase-9 in the synovial tissue. In conclusion, Col V supplementation decreased synovial inflammation and the fibrotic response, possibly by increased the apoptosis of inflammatory cells.
Assuntos
Artrite Experimental/tratamento farmacológico , Colágeno Tipo V/farmacologia , Membrana Sinovial/efeitos dos fármacos , Administração Oral , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Caspase 9/metabolismo , Citocinas/metabolismo , Edema/tratamento farmacológico , Edema/imunologia , Edema/patologia , Adjuvante de Freund , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Ratos Endogâmicos Lew , Soroalbumina Bovina , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
BACKGROUND: Alternative splicing of EDA fibronectin (FN) region is a cell type- and development-regulated mechanism controlled by pathological processes, growth factors and extracellular matrix (ECM). Classic and vascular Ehlers-Danlos syndrome (cEDS and vEDS) are connective tissue disorders caused by COL5A1/COL5A2 and COL3A1 gene mutations, leading to an in vivo abnormal collagen fibrillogenesis and to an in vitro defective organisation in the ECM of type V (COLLV) and type III collagen (COLLIII). These defects induce the FN-ECM disarray and the decrease of COLLs and FN receptors, the α2ß1 and α5ß1 integrins. Purified COLLV and COLLIII restore the COLL-FN-ECMs in both EDS cell strains. METHODS: Real-time PCR, immunofluorescence microscopy, and Western blotting were used to investigate the effects of COLLs on FN1 gene expression, EDA region alternative splicing, EDA(+)-FN-ECM assembly, α5ß1 integrin and EDA(+)-FN-specific α9 integrin subunit organisation, α5ß1 integrin and FAK co-regulation in EDS fibroblasts. RESULTS: COLLV-treated cEDS and COLLIII-treated vEDS fibroblasts up-regulate the FN1 gene expression, modulate the EDA(+) mRNA maturation and increase the EDA(+)-FN levels, thus restoring a control-like FN-ECM, which elicits the EDA(+)-FN-specific α9ß1 integrin organisation, recruits the α5ß1 integrin and switches on the FAK binding and phosphorylation. CONCLUSION: COLLs regulate the EDA(+)-FN-ECM organisation at transcriptional and post-transcriptional level and activate the α5ß1-FAK complexes. COLLs also recruit the α9ß1 integrin involved in the assembly of the EDA(+)-FN-ECM in EDS cells. GENERAL SIGNIFICANCE: The knowledge of the COLLs-ECM role in FN isotype expression and in EDA(+)-FN-ECM-mediated signal transduction adds insights in the ECM remodelling mechanisms in EDS cells.
Assuntos
Colágeno Tipo III/farmacologia , Colágeno Tipo V/farmacologia , Síndrome de Ehlers-Danlos/patologia , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacosRESUMO
Type V collagen is known to be over-deposited in the stroma of ductal infiltrating carcinomas of the breast. When used as a substrate, type V collagen restrains growth and invasion, and affects gene expression of 8701-BC ductal infiltrating carcinomas cells. Here we supplement existing data by demonstrating type V collagen dependent upregulation of capn2 gene expression in 8701-BC cells through differential display-PCR and Western blot assays. Furthermore, we suggest that our data obtained by centrifugal sedimentation and electrophoresis strongly suggest a correlation between calpain overproduction and DNA fragmentation, since the incubation with calpain inhibitor partly reverts the latter.
Assuntos
Neoplasias da Mama/enzimologia , Calpaína/genética , Colágeno Tipo V/farmacologia , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Reação em Cadeia da PolimeraseRESUMO
Primary rat hepatocytes were cultured on type V collagen. Hepatocyte spreading on type V collagen was inferior compared with that on type I collagen. However, the albumin production rates of hepatocytes cultured on type V collagen were approximately twice as high as those of hepatocytes cultured on type I collagen.
Assuntos
Albuminas/biossíntese , Colágeno Tipo V/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Colágeno Tipo I/farmacologia , Ensaio de Imunoadsorção Enzimática , RatosRESUMO
Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present.
Assuntos
Colágeno Tipo V/metabolismo , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Adesões Focais , Actinas/metabolismo , Movimento Celular , Forma Celular , Colágeno Tipo V/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Fibroblastos/efeitos dos fármacos , Humanos , Hidrogéis/metabolismoRESUMO
Mutations in the genes encoding for type V collagen have been found in the classical type of Ehlers-Danlos syndrome (EDS); the most common mutations lead to a non-functional COL5A1 allele. We characterized three skin fibroblast strains derived from patients affected by classical EDS caused by COL5A1 haploinsufficiency. As a typical clinical hallmark of EDS is the impaired wound healing, we analyzed the repair capability of fibroblasts in a monolayer wounding assay. The mutant fibroblast strains were unable to move into the scraped area showing then a marked delay in wound repair. In all the EDS strains, type V collagen was absent in the extracellular space, also leading to the lack of fibronectin fibrillar network and impairing the expression of alpha(2)beta(1) and alpha(5)beta(1) integrins. The abnormal integrin pattern inhibited the positive effect of insulin-like growth factor-binding protein-1 on cell migration, whereas the migratory capability remarkably improved in the presence of exogenous type V collagen.
Assuntos
Movimento Celular/fisiologia , Colágeno Tipo V/farmacologia , Síndrome de Ehlers-Danlos/patologia , Fibroblastos/patologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Alelos , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Integrina alfa5beta1/metabolismo , Mutação/genética , Cicatrização/fisiologiaRESUMO
The third alpha-chain of type V collagen, alpha3(V) chain, was initially identified in the placenta more than 20 years ago, but was poorly characterized with regard to its expression and function. We generated a specific monoclonal antibody against the N-terminal domain of the pro-alpha3(V) chain and examined gene expression using immunohistochemical methods combined with in situ hybridization. The pro-alpha3(V) chain was seen in funis and amnion, but not chorionic villi and deciduas of mouse placenta. In mouse embryo, the transcripts of the pro-alpha3(V) gene were seen in tissues that were related to bone formation as well as developing muscle and nascent ligament previously reported. However, immunohistochemistry showed that pro-alpha3(V) protein accumulated rather in the developing bone of mouse embryo. On the other hand, the N-terminal globular domain of the pro-alpha3(V) chain has a unique structure that contains a highly basic segment of 23 amino acids. The peptide derived from the basic segment showed a specific adhesive feature to osteosarcoma cells but not to chondrosarcoma cells. The four heparin binding sites in the basic segment equally contribute toward adhesion to the osteosarcoma cells. Our data suggested that N-terminal globular domain of the pro-alpha3(V) chain influence bone formation of osteoblasts through proteoglycan on the cell surface during development or regeneration.
Assuntos
Osso e Ossos/metabolismo , Colágeno Tipo V/química , Colágeno Tipo V/metabolismo , Regulação da Expressão Gênica , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Colágeno Tipo V/genética , Colágeno Tipo V/farmacologia , Embrião de Mamíferos/metabolismo , Heparina/química , Heparina/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Osteossarcoma/genética , Placenta/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , RatosRESUMO
The airways of patients with chronic asthma commonly develop an element of fixed airway obstruction, which fails to reverse with inhaled beta2-adrenoceptor agonists. Airway remodeling refers to the structural changes of the bronchi in longstanding asthma and is characterized by increased deposition and altered ratios of extracellular matrix (ECM) proteins. We therefore assessed whether ECM proteins alter beta2-adrenoceptor signaling in human airway smooth muscle cells. We report that a fibronectin environment increases responses to beta2-adrenoceptor stimulation, whereas exposure to collagen V or laminin decreases accumulation of the second messenger cyclic AMP when compared with collagens I or IV. These differences are likely to be physiologically significant as they translate into altered phosphorylation of the downstream target VASP. The altered cAMP levels are due to differences in adenylyl cyclase activity, although expression of the relevant isoforms of enzyme appears unaltered. However, inhibition of Galphai abrogates the differences in beta2-adrenoceptor-mediated cAMP accumulation in cells exposed to different matrix factors. The difference in Galphai signaling is not due to altered Galphai expression. We conclude therefore that ECM modulates Galphai activity in human airway smooth muscle cells, and propose that these changes could contribute to the fixed airway obstruction seen in patients with chronic asthma.
Assuntos
Obstrução das Vias Respiratórias , Asma/fisiopatologia , Matriz Extracelular/fisiologia , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Adenilil Ciclases/metabolismo , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/fisiopatologia , Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Moléculas de Adesão Celular/metabolismo , Colágeno Tipo V/farmacologia , AMP Cíclico/metabolismo , Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/farmacologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Laminina/farmacologia , Proteínas dos Microfilamentos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/químicaRESUMO
In the present study we investigated whether the collagen types I, III and V affect the activity of fibroblasts obtained from rabbit periosteum. The cells were cultured on plates either or not coated with different amounts of collagen type I, III or V and analyzed for their attachment, DNA synthesis and the expression and activity of matrix metalloproteinases (MMPs). Our data show that the three collagen types promoted attachment and spreading of the cells and stimulated DNA synthesis when used in relatively low concentrations. High concentrations of type V-but not of type I or III-proved to inhibit thymidine incorporation. The expression and activity of matrix metalloproteinase 1 (MMP-1; interstitial collagenase) decreased under the influence of relatively low amounts of collagen (<40 microg/well), whereas higher levels increased its release. Matrix metalloproteinase 2 (MMP-2; gelatinase A) was up-regulated by the different types of collagen; the active fraction of stromelysin-1 (MMP-3) decreased. Accordingly, the mRNA expression of MMP-1 and -3 were reduced. The expression of MMP-2 mRNA, however, proved to be unaffected. Blocking antibodies to beta(1)-integrin or echistatin increased the level of MMP-1 but had no effect on MMP-2. All parameters tested were similarly affected by type I and III collagen, whereas the effect of type V was always less. We conclude that the collagen types I, III and V provide different sets of signals for fibroblasts that differently modulate their proliferation and MMP expression.
Assuntos
Colágeno/farmacologia , Metaloproteinases da Matriz/metabolismo , Animais , Sequência de Bases , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/farmacologia , Colágeno Tipo II/metabolismo , Colágeno Tipo II/farmacologia , Colágeno Tipo V/metabolismo , Colágeno Tipo V/farmacologia , DNA/biossíntese , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinases da Matriz/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , CoelhosRESUMO
Airway remodeling complicates longstanding asthma. It is characterized by an increase in the number of airway smooth muscle cells (SMCs) as well as an increase in and alteration of the type of extra-cellular matrix (ECM) in the airways. Although the number of SMCs in the airways depends on the balance of cell proliferation and cell death, studies to date have concentrated on factors affecting SMC proliferation. Here we report the first study on airway SMC survival factors: these cells receive a strong survival signal, which is not dependent on the known growth factor mitogens. We identified the ECM factors fibronectin, laminin, and collagens I and IV as important anti-apoptotic elements, and characterized the expression of the ECM receptors (integrins) on cultured SMC. Functionally blocking antibody and peptide studies revealed the alpha(5)beta(1) integrin to be an important transducer of the ECM-derived survival signal in these cells. Confocal microscopy confirmed the striking effects that discrete ECM factors have on SMC phenotype, notably the cytoskeleton. In summary, our data improves the understanding of the mechanisms underlying airway remodeling by outlining the key survival factors for airway SMC and by highlighting the impact of the cell-matrix interactions on cell death and phenotype.
Assuntos
Proteínas da Matriz Extracelular/farmacologia , Substâncias de Crescimento/farmacologia , Músculo Liso/citologia , Traqueia/citologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Colágeno Tipo I/farmacologia , Colágeno Tipo IV/farmacologia , Colágeno Tipo V/farmacologia , Citocinas/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Elastina/farmacologia , Fibroblastos/química , Fibroblastos/citologia , Fibronectinas/farmacologia , Humanos , Laminina/farmacologia , Receptores de Fibronectina/análise , Receptores de Fibronectina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vitronectina/farmacologiaRESUMO
The contents of type I, type III and type V collagen and the collagen type specific distributions in liver under normal and cirrhotic conditions were examined. In CCl4 injected rat, the increasing amount of type V collagen was a specific event during the progression of cirrhosis. In normal liver, immunohistochemical observation showed that type V collagen was localized on the fine fibrils, while type I was localized on the thick fibril. Type V collagen was partially colocalized with type IV collagen. In the cirrhotic liver, type V collagen was localized on the margin of the thick fibrous septa along with type IV collagen. Type I collagen existed in the core region of fibrous septa where the stellate cells were prominent. To elucidate the mechanism of the type specific deposition of collagen in the liver, we constructed a coculture system using both stellate cells and hepatocytes. In this system, type V collagen was mainly deposited on hepatocyte colonies not on stellate cells, while type I collagen fibrils were localized on stellate cells. The spatial positioning of type I and type V collagens in vitro was similar to that in the liver. In the cell adhesion assay, the adhesion of stellate cells to type V collagen was poorer than that of the hepatocytes. The collagen type-specific affinity of the stellate cells and hepatocytes may explain the specific localization of type V collagen in the liver and coculture system. These results suggested that the functions of type V collagen are not only to connect type IV collagen with type I collagen fibril, but also to protect the parenchyma from excess type I collagen deposition produced by stellate cells under pathological conditions.