RESUMO
The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvß3 and αvß5 significantly change the biological activities against αvß6 and αvß8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).
Assuntos
Fibrose Pulmonar Idiopática/patologia , Integrina alfaV/química , Fenilbutiratos/química , Administração Oral , Animais , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Integrina alfaV/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Fenilbutiratos/farmacocinética , Fenilbutiratos/uso terapêutico , Estrutura Terciária de Proteína , Ratos , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Relação Estrutura-AtividadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (CR) has been traditionally used as an herbal medicine in Asian countries to treat diverse gynecological disorders. However, the potential therapeutic effect of CR on endometrial receptivity for successful embryo implantation to treat female infertility has not been fully studied. AIM OF STUDY: The aim of this study was to evaluate the effect of water-extracted CR on endometrial receptivity by investigating the expression of leukemia inhibitory factor (LIF) and integrins, cell adhesion, and embryo implantation using mifepristone (RU486; RU)-induced implantation failure model. MATERIALS AND METHODS: The water extract of CR was prepared and fingerprinted using high-performance liquid chromatography (HPLC). For the expression and regulation of LIF, reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed in CR-stimulated Ishikawa cells. To evaluate LIF-mediated integrin expression, knockdown of LIF by shRNA was performed in Ishikawa cells. The effect of CR on endometrial receptivity was determined by an in vitro adhesion assay between JAr cells and CR-induced Ishikawa cells. In vivo, C57BL/6 female mice (n = 7 per group) orally received CR (31.68mg/kg/day), a similar dose as used clinically. Seven days after CR treatment, all female mice were caged with male mice until pregnancy was verified. On day 4 of pregnancy, RU (4mg/kg) was injected subcutaneously to induce embryo implantation failure. RESULT: CR increased the expression of LIF through the phosphatidylinositol-3-kinase/ protein kinase B (PI-3K/AKT) signaling pathway in Ishikawa cells. In addition, CR enhanced adhesion of JAr cells onto Ishikawa cells by inducing the expression of LIF-dependent integrins αVß3 and αVß5. Furthermore, CR improved the number of implantation sites in pregnant mice despite RU injection. CONCLUSION: CR increased the expression of LIF-mediated integrins αVß3 and αVß5 on the surface of endometrial cells, which is associated with adhesion of trophoblastic cells to endometrial cells for blastocyst implantation. Our findings provide evidence that CR has therapeutic potential against poor endometrial receptivity.
Assuntos
Cyperus , Implantação do Embrião/efeitos dos fármacos , Integrina alfaVbeta3/metabolismo , Fator Inibidor de Leucemia/metabolismo , Extratos Vegetais/farmacologia , Receptores de Vitronectina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Feminino , Humanos , Integrina alfaVbeta3/genética , Fator Inibidor de Leucemia/genética , Masculino , Camundongos Endogâmicos C57BL , Mifepristona/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Tubérculos/química , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Vitronectina/genética , Água/químicaRESUMO
BACKGROUND: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) has been attributed to increased systemic inflammation and insulin resistance mediated by visceral adipose tissue (VAT), although the exact mechanisms are undefined. Exosomes are membrane-derived vesicles containing messenger RNA, microRNA, and proteins, which have been implicated in cancer, neurodegenerative, and autoimmune diseases, which we postulated may be involved in obesity-related diseases. We isolated exosomes from VAT, characterized their content, and identified their potential targets. Targets included the transforming growth factor beta (TGF-ß) pathway, which has been linked to NAFLD. We hypothesized that adipocyte exosomes would integrate into HepG2 and hepatic stellate cell lines and cause dysregulation of the TGF-ß pathway. METHODS: Exosomes from VAT from obese and lean patients were isolated and fluorescently labeled, then applied to cultured hepatic cell lines. After incubation, culture slides were imaged to detect exosome uptake. In separate experiments, exosomes were applied to cultured cells and incubated 48-h. Gene expression of TGF-ß pathway mediators was analyzed by polymerase chain reaction, and compared with cells, which were not exposed to exosomes. RESULTS: Fluorescent-labeled exosomes integrated into both cell types and deposited in a perinuclear distribution. Exosome exposure caused increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and integrin ανß-5 expression and decreased matrix metalloproteinase-7 and plasminogen activator inhibitor-1 expression in to HepG2 cells and increased expression of TIMP-1, TIMP-4, Smad-3, integrins ανß-5 and ανß-8, and matrix metalloproteinase-9 in hepatic stellate cells. CONCLUSIONS: Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-ß pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD.
Assuntos
Adipócitos/metabolismo , Exossomos/metabolismo , Hepatócitos/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adipócitos/patologia , Adolescente , Feminino , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/patologia , Humanos , Integrinas/metabolismo , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Metaloproteinase 7 da Matriz/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/patologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Pancreáticas/terapia , Compostos Radiofarmacêuticos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos Monoclonais/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Radioisótopos do Iodo , Camundongos , Camundongos Nus , Pâncreas/imunologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Ácido Pentético/análogos & derivados , Ácido Pentético/farmacocinética , Radioimunoensaio , Receptores de Vitronectina/imunologia , Receptores de Vitronectina/metabolismo , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Integrins are transmembrane proteins that facilitate the interaction of cells with the extracellular environment. They have also been implicated in cancer progression. The effects of nutrients thought to be involved in the prevention of prostate cancer on integrin expression have not been determined. Prostate cancer cell lines representing a range of malignancy from normal (RWPE-1) to highly invasive phenotypes (22Rv1 < LNCaP < PC-3) were cultured with or without lycopene (10 nM), vitamin E (5 microm) or fish oil (100 microm) for 48 h. Growth and integrin (alpha2beta1, alphavbeta3 and alphavbeta5) expression were assessed using Trypan Blue exclusion and monoclonal antibodies combined with flow cytometry. Vitamin E enhanced (P < 0.001) whereas fish oil reduced the growth of all the cell lines tested (P < 0.001). Lycopene had no effect on growth. All the malignant cell lines exhibited lower expression of alpha2beta1 with the addition of lycopene to culture media. Supplemental fish oil reduced alpha2beta1 in most invasive cell lines (LNCaP and PC-3). Each nutrient at physiological levels reduced integrins alphavbeta3 and alphavbeta5 in most invasive cell lines (PC-3). The results suggest that integrins may represent an additional target of bioactive nutrients and that the effects of nutrients may be dependent on the type of cell line used.
Assuntos
Carotenoides/farmacologia , Óleos de Peixe/farmacologia , Integrinas/metabolismo , Neoplasias da Próstata/metabolismo , Vitamina E/farmacologia , Animais , Carotenoides/metabolismo , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Depressão Química , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Sangue Fetal/metabolismo , Óleos de Peixe/metabolismo , Humanos , Integrina alfa2beta1/análise , Integrina alfa2beta1/metabolismo , Integrina alfaVbeta3/análise , Integrina alfaVbeta3/metabolismo , Integrinas/análise , Licopeno , Masculino , Próstata/química , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/química , Neoplasias da Próstata/patologia , Receptores de Vitronectina/análise , Receptores de Vitronectina/metabolismo , Vitamina E/metabolismoRESUMO
3-(S)-Pyrimidin-5-yl-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5e) and 3-(S)-(methylpyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5f) were identified as potent and selective antagonists of the alpha(v)beta(3) receptor. These compounds have excellent in vitro profiles (IC(50) = 0.07 and 0.08 nM, respectively), significant unbound fractions in human plasma (6 and 4%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in an in vivo model of bone turnover following once-daily oral administration, these two compounds were selected for clinical development for the treatment of osteoporosis.
Assuntos
Integrinas/antagonistas & inibidores , Naftiridinas/farmacologia , Osteoporose/tratamento farmacológico , Receptores de Vitronectina/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Humanos , Concentração Inibidora 50 , Integrinas/metabolismo , Macaca mulatta , Modelos Moleculares , Naftiridinas/química , Naftiridinas/farmacocinética , Osteoporose/prevenção & controle , Ovariectomia , Ratos , Ratos Sprague-Dawley , Receptores de Vitronectina/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements.
Assuntos
Aminoácidos/química , Ésteres/química , Receptores de Vitronectina/antagonistas & inibidores , Aminoácidos/síntese química , Aminoácidos/farmacocinética , Animais , Ácido Aspártico/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacocinética , Cães , Avaliação Pré-Clínica de Medicamentos , Ésteres/síntese química , Ésteres/farmacocinética , Humanos , Concentração Inibidora 50 , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Ligação Proteica , Receptores de Vitronectina/metabolismo , Relação Estrutura-AtividadeRESUMO
Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for cancer. However, a primary limiting factor for the use of Ad vectors for cancer gene therapy is their critical dependence on cellular expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. Following the identification of CAR deficiency in RCC lines, we have found abundant membrane expression of alpha(v)beta 3 and alpha(v)beta 5 integrins and of the putative receptor to Ad serotype 3 (Ad3). As an alternative gene therapy approach for RCC that would circumvent CAR deficiency, we employed retargeting of replication-incompetent Ad vectors and replication-competent Ad viruses to alpha(v)beta 3 and alpha(v)beta 5 integrins and to the putative Ad3 receptor. These strategies to genetically alter Ad tropism were based on either the insertion of a cysteine-aspartate-cysteine-arginine-glycine-aspartate-cysteine-phenylalanine-cysteine (RGD) motif into the HI loop of the Ad fiber knob domain or on generation of a chimeric Ad fiber composed of adenovirus serotype 5 shaft/Ad3 knob. Both strategies proved highly efficient to circumvent CAR deficiency and enhance gene delivery into RCC cells. Furthermore, in the context of replication-competent Ad, tropism alteration resulted in distinct capacity of the retargeted viruses to infect, replicate, and lyse RCC models in vitro and in vivo. The retargeting strategies were particularly beneficial in the context of replication-competent Ad. These findings underscore the importance of CAR-independent cellular entry mechanisms in RCC and are highly consequential for the development of viral antitumor agents for RCC and other CAR-negative tumors.
Assuntos
Adenoviridae/genética , Carcinoma de Células Renais/terapia , Terapia Genética/métodos , Integrinas/metabolismo , Neoplasias Renais/terapia , Receptores de Vitronectina/metabolismo , Adenoviridae/metabolismo , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/virologia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/virologia , Camundongos , Camundongos Nus , Receptores Virais/deficiência , Receptores Virais/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mimetics of the RGD tripeptide are described that are potent, selective antagonists of the integrin receptor, alpha(v)beta(3). The use of the 5,6,7,8-tetrahydro[1,8]naphthyridine group as a potency-enhancing N-terminus is demonstrated. Two 3-substituted-3-amino-propionic acids previously contained in alpha(IIb)beta(3) antagonists were utilized to enhance binding affinity and functional activity for the targeted receptor. Further affinity increases were then achieved through the use of cyclic glycyl amide bond constraints.
Assuntos
Oligopeptídeos/farmacologia , Receptores de Vitronectina/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Ligação Competitiva , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Conformação Molecular , Mimetismo Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Osteoporose/prevenção & controle , Agregação Plaquetária/efeitos dos fármacos , Receptores de Vitronectina/metabolismo , Relação Estrutura-AtividadeRESUMO
Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.
Assuntos
Oligopeptídeos/farmacocinética , Receptores de Vitronectina/antagonistas & inibidores , Aminoácidos/química , Animais , Ácido Aspártico , Ligação Competitiva , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Mimetismo Molecular , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Osteoporose/prevenção & controle , Ligação Proteica , Receptores de Vitronectina/metabolismo , Relação Estrutura-AtividadeRESUMO
Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C-D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.
Assuntos
Apoptose , Artrite Experimental/terapia , Terapia Genética/métodos , Neovascularização Patológica/terapia , Oligopeptídeos/farmacologia , Membrana Sinovial/irrigação sanguínea , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Bacteriófago M13/genética , Ligação Competitiva , Colágeno , Sistemas de Liberação de Medicamentos/métodos , Marcação In Situ das Extremidades Cortadas , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Neovascularização Patológica/patologia , Fragmentos de Peptídeos/farmacologia , Receptores de Vitronectina/metabolismo , Membrana Sinovial/imunologiaRESUMO
In attempt to find novel integrin alphavbeta3 antagonists, we selected SC65811 and its guanidine analogue (1) as lead compounds. Modification of the glycine part of SC65811 led to a new series of malonamide derivatives that exhibited alphavbeta3 inhibitory activity. Among them, (R,S)-3-[3-[6-(3-benzylureido)indolin-1-yl]-3-oxopropanoylamino]-3- (pyridin-3-yl)propanoic acid (43a) showed not only potent activity with an IC50 value of 3.0 nM but also good selectivity for alphavbeta3 relative to alphaIIbbeta3, alpha5beta1, and alphavbeta5 with IC50 values of 19,000, 11,000, and 14 nM, respectively. Furthermore, optimization of 43a led to the most potent alphavbeta3 antagonist, (R,S)-3-(3-[6-[(4,5-dihydro-1H-imidazol-2-yl)amino]indolin-1-yl]-3-oxopropanoylamino)-3-(quinolin-3-yl)propanoic acid (431) with an IC50 value of 0.42 nM. The synthesis and the structure-activity relationships of these malonamide derivatives are presented.
Assuntos
Malonatos/síntese química , Malonatos/metabolismo , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Compostos de Anilina/síntese química , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Malonatos/química , Relação Estrutura-AtividadeRESUMO
Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors alpha 5 beta 1 and alpha v beta 3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of alpha 5 beta 1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by alpha 5 beta1 integrin ligation is mediated via induction and autocrine production of IL-1 beta, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, alpha v beta 3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1 beta, NO, and PGE2 (triggered by alpha 5 beta 1, IL-1 beta, IL-18, or IL-1 beta, TNF-alpha, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated "outside in" and "inside out" signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.
Assuntos
Cartilagem Articular/imunologia , Mediadores da Inflamação/metabolismo , Osteoartrite/genética , Osteoartrite/imunologia , Receptores de Fibronectina/fisiologia , Receptores de Vitronectina/fisiologia , Adulto , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Bovinos , Condrócitos/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Humanos , Interleucina-1/antagonistas & inibidores , Interleucina-1/biossíntese , Interleucina-1/genética , Interleucina-18/fisiologia , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Ligantes , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Pessoa de Meia-Idade , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Osteoartrite/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fibronectina/antagonistas & inibidores , Receptores de Fibronectina/imunologia , Receptores de Fibronectina/metabolismo , Receptores de Vitronectina/imunologia , Receptores de Vitronectina/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Regulação para Cima/imunologiaRESUMO
We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.
Assuntos
Osso e Ossos/metabolismo , Substâncias de Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Neoplasias , Osteoblastos/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteínas de Sinalização Intercelular CCN , Adesão Celular , Clonagem Molecular , DNA Complementar/genética , Fibrinogênio/metabolismo , Substâncias de Crescimento/genética , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica , Osteocalcina/biossíntese , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Ratos , Receptores de Vitronectina/metabolismo , Proteínas Repressoras , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the alphavbeta3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the alphavbeta3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.