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BACKGROUND:Transglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domains] within the protein.METHODS:Human prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.RESULTS:TGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.CONCLUSION:TGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

The rate limiting step of phagocytosis is the binding of collagen to specific receptors, which include β1 integrins and the discoidin domain receptor 1 (DDR1). While these two receptors may interact, the functional nature of these interactions is not defined. We examined the effects of DDR1 over-expression on β1 integrin function and determined that DDR1 over-expression enhanced cell attachment through β1 integrins. These data are consistent with data showing that DDR1 over-expression enhanced cell-surface, but not total, β1 integrin expression and activation. As shown by experiments with endoglycosidase H, DDR1 over-expression increased glycosylation of the β1 integrin subunit. Collectively these data indicate that DDR1 enhances β1 integrin interactions with fibrillar collagen, possibly by affecting the processing and trafficking of β1 integrins to the cell surface. Our data provide insight into the mechanisms by which fibrotic conditions such as cyclosporine A-induced gingival overgrowth are regulated.

During cardiac development and in cardiac disease changes in hemodynamic load initiate events leading to remodeling of the ECM. This study addresses the hypothesis that interactions between Integrins and Metalloprotienases function to modulate cell adhesion in the cultured cardiac fibroblast. The fibroblast is positioned to detect and respond to changes in the mechanical load on the heart. Functionally the cardiac fibroblast is the primary cell type responsible for the production, maintenance, and remodeling of the cardiac interstitium. Matrix Metalloproteinases, specifically the Gelatinases, are expressed in concert during development and in disease with changes in the hemodynamic loading of the heart. Our studies have identified by a complex on the surface of the cardiac fibroblast composed of the a3b1 integrin, MMP-2, and TIMP-2. Putatively, this complex is involved in the maturation of adhesions. Inhibition of MMP-2 was associated with a decrease in the strength of adhesion of cell plated on collagen and fibronectin. Confocal imaging and analysis indicate that a predominate interaction occurs between MMP-2 and the a3 integrin chain. Taken together biochemical, functional, and microscopic data have identified a complex on the surface of the cardiac fibroblast that represents elements of mechanotransduction and matrix metabolism in a single site that functions in the maturation of adhesion

Introdução: In vivo ou in vitro a exposição de fibroblastos a alta concentração de glicose promove um aumento do estresse oxidativo e consequentemente prejudica a migração celular, assim como a maturação da adesão. Além disso, a elevada concentração de glicose reduz a expressão de diferentes integrinas na superfície celular devido alterações na síntese do receptor e sua reciclagem. Objetivo: Avaliar os efeitos da elevada concentração de glicose no tráfego de vesículas contendo EEA1 (endossomos primários), Rab4 (via rápida da reciclagem), Rab11 (via lenta de reciclagem) e Rab7 (endossomos de degradação) em fibroblastos NIH3T3. Métodos: células foram cultivadas em meio contendo baixa concentração de glicose (LG, 5 mM) ou em alta concentração (HG 25 mM) durante 21 dias antes de realizar os experimentos. EEA1, Rab4, Rab11 e Rab7 expressão e distribuição foram avaliados por western blotting e imunofluorescência, respectivamente. Resultados: Células expostas à alta concentração não apresentaram diferenças na expressão e distribuição das proteínas EEA1 e Rab7, enquanto a expressão de Rab11 foi reduzida em 30%. Conclusão: a alta concentração de glicose altera a via lenta da reciclagem contendo Rab11, afetando potencialmente a reciclagem de integrinas e outros receptores e a sua expressão na superfície celular. / Background: In vivo or in vitro exposure of fibroblasts to high glucose concentrations (HG) promotes oxidative stress and consequently impairs cell migration, also inhibiting adhesion maturation. Additionally, HG reduces the expression of different integrins on the cell surface, potentially due to altered receptor synthesis and recycling. Aim: to evaluate the effects of HG on the trafficking vesicles containing EEA1 (early endosomes), Rab4 (fast recycling pathway) and Rab7 (endocytic degradation pathway) on NIH3T3 fibroblasts. Methods: cells were cultured under low glucose (LG, 5 mM) or HG (25 mM) concentrations during 21 days before the assays. EEA1, Rab4 and Rab7 expression and distribution were evaluated by western blotting and immunofluorescence, respectively. Results: HG did not affect proteins EEA1 and Rab7 expression and distribution, whereas Rab11 expression was reduced by 30%. The number of vesicles containing Rab11 was also significantly reduced in HG cells. Conclusion: high glucose alters the slow recycling endocytic pathway via Rab11, potentially affecting integrins and other receptors synthesis and expression on the cell surface.

Diversos estudos, in vivo e in vitro, demonstraram a atividade antitumoral da Crotoxina (CTX), toxina majoritária do veneno de serpente Crotalus durissus terrificus, porém, não foi demonstrada, até o momento, a ação desta toxina sobre eventos envolvidos com a neovascularização, essenciais para o crescimento e sobrevivência do tumor. Assim, neste estudo foi investigado o efeito in vitro da CTX sobre os eventos envolvidos com a angiogênese. A CTX inibiu, principalmente na concentração de 1,2µg/mL, a proliferação, adesão e a morfologia das células endoteliais murinas derivados do hemangioma do timo (t.End.1) sobre as matrizes de laminina, colágeno tipo I e fibronectina, bem como a migração por meio dos modelos de cicatrização (Wound Healing), quimiotaxia (transwell) e a formação de tubos no matrigel 3-D, tanto na presença de meio de cultura como no meio condicionado de célula tumoral. Ainda, a CTX inibiu a distribuição das subunidades v e 2 de integrinas e a polimerização do citoesqueleto de actina, evidenciados em microscopia confocal. Os resultados demonstram, pela primeira vez, que a CTX inibe os principais eventos envolvidos com a angiogênese, podendo contribuir de forma importante para o efeito inibitório da toxina sobre a progressão tumoral. / Several studies, in vivo and in vitro have demonstrated antitumor activity of Crotoxin (CTX), the major toxin of Crotalus durissus terrificus venom. Despite evidence of antitumor action of CTX, was not demonstrated yet the action of this toxin on basic parameters for neovascularization, essential for the growth and survival of the tumor. The formation of new blood vessels is the principal process of angiogenesis and involves adhesion, proliferation and migration of endothelial cells to reach remote targets, assembly of endothelial cells into new capillary tubes. CTX (1.2 µg/mL, for 1 hour incubation), inhibited cell proliferation, adhesion, migration, scratch wound healing and capillary-like tube formation on 3-D matrix of endothelial cells line derived from thymus (t.End.1) evaluated in vitro assay at culture medium or conditioned medium obtained from tumour cells culture. Also, this toxin interfered with the distribution of the integrin subunits 2 and v and with the cytoskeleton rearrangement these cells, evidenced in confocal microscope. Taken together, these results demonstrated for the first time, that CTX inhibits key events involved in the angiogenesis process, and may contribute for inhibitory effect of the toxin on tumor progression.

Plaquetas e células tumorais interagem em uma reação cruzada com proteínas do plasma, via integrina αIIbβ3 e αvβ3, respectivamente. A integrina αvβ3 também encontra-se presente na angiogênese tumoral. O objetivo desse trabalho foi avaliar a GST-INS, uma disintegrina recombinante do veneno de Bothrops insularis em eventos da progressão tumoral. Em condições estáticas, GST-INS foi capaz de inibir totalmente a adesão de células HUVECs e SK-MEL-28 às plaquetas em comparação ao controle e ao Aggrastat® (inibidor seletivo da integrina αIIbβ3). Além de inibir a TCIPA (agregação plaquetária induzida por células tumorais) a GST-INS também inibiu a invasão de SK-MEL-28 em substrato de matrigel. Células t.End.1 ou SK-MEL-28 pré-incubadas com GST-INS não formaram túbulos no substrato de matrigel. Análise por microscopia confocal mostrou que GST-INS liga-se a integrina αv presente nas células SK-MEL-28. Nossos resultados sugerem que essa disintegrina pode ser utilizada como potencial ferramenta no estudo e desenvolvimento de antiangiogênicos e antimetastáticos. / Platelets and tumor cells interact in a cross-react with plasma proteins via integrin αIIbβ3 and αvβ3 , respectively.The integrin αvβ3 is also strongly stimulated in tumor angiogenesis. The aim of this study was to evaluate the ability of GST-INS, a recombinant disintegrin from Bothrops insularis venom on events of tumor progression. Under static conditions, GST-INS was able to completely inhibit the adhesion of endothelial cells (HUVECs) and melanoma cells (SK-MEL-28) to platelets compared to control and Aggrastat® (selective inhibitor of integrin αIIbβ3). In addition, GST-INS inhibit TCIPA (platelet aggregation induced by tumor cells) GST-INS also inhibited SK-MEL-28 on matrigel invasion substrate. t.End.1 cells or SK-MEL-28 pre-incubated with GST-INS were not able to form tubules in matrigel substrate. Analysis by confocal microscopy showed that GST-INS binds to integrin αv present in SK-MEL-28 cells. The results suggest that disintegrin can be used as a potential tool in the study and development of antiangiogenic and antimetastatic.

Tumor cells interact with basement membrane collagen at the site of extravasation through distinct cellular receptors, including the α2β1 and α3β1integrins. These receptors are known to be differentially expressed in metastatic tumors, relative to the normal cells, depending on tumor type and stage of progression. The binding sites within type IV collagen for the α2β1 andα3β1 integrins have been identified. Since both of the integinspecific sequences possess at least one glycosylated Hyl residue, we questioned whether glycosylation could modulate integrin binding. Triple-helical peptides with and without Lys substituted by glycosylated Hyl for Lys543 and Lys540 from the human a1(IV)531-543 gene sequence (α3β integrin-specific) and Lys393 from the human a1(IV)382-393 gene sequence (α2β1 integrin-specific) were synthesized and utilized in the present study. / Cellular response to these triple helical ligands was tested with a primary melanoma cell line, WM-115, and three highly metastatic melanoma cell lines , WM-266-4, M14#5, and SK-MEL-2. Cell adhesion and cell spreading assays yielded differing results depending on whether the ligands contained glycosylated Hyl residues or not. In general, a decrease in cellular affinity toward the ligands was observed when glycosylated Hyl was present. Differences in the levels of adhesion and spreading between cell lines representing different stages of melanoma were also observed. Neutral B-galactosidase activity was detected in all four cell lines. Enzymatic activity levels were comparable for the three metastatic cell lines, whereas distinctively higher activity was detected for cells originating from a primary lesion. This acitivity can signal the potential of tumor cells to enhance and recover their invasive abilities. / The ability of each cell line to remove the galactose from the peptide ligands has been investigated, to test whether tumor cells can reestablish binding relationships between the α2β1 and α3β1 integrins and type IV collagen that are reduced by glycosylation. / by Beatrix Aukszi. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.