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Alternagin-C binding to ?2?1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

ABSTRACT: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of ?2?1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an ?2?1 integrin. Herein, we used ALT-C as a ?2?1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration.ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The ?2?1 integrin binding properties of ALT-C, its dissociation constant (Kd ) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting.Our data demonstrate that ALT-C, after binding to ?2?1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression.These results demonstrate that ?2?1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.

SUBMITTER: Moritz MNO

PROVIDER: S-EPMC5917863 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Alternagin-C binding to α<sub>2</sub>β<sub>1</sub> integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

Moritz Milene Nóbrega de Oliveira MNO Eustáquio Lívia Mara Santos LMS Micocci Kelli Cristina KC Nunes Ana Carolina Caetano ACC Dos Santos Patty Karina PK de Castro Vieira Tamires T Selistre-de-Araujo Heloísa Sobreiro HS

The journal of venomous animals and toxins including tropical diseases 20180425

<h4>Background</h4>Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major c ...[more]

PMID: 29713337

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Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin ?5/?1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin ?5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin ?5/?1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin ?5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin ?5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and ?-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3?, and p-GSK-3? were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin ?5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin ?5 antibody groups. At the same time, expression of GSK-3? and p-GSK-3? was upregulated and ?-catenin levels showed no difference between the groups with or without anti-integrin ?5 antibody. Since Akt, p-Akt, GSK-3?, and p-GSK-3? displayed the same changes between the groups with or without anti-integrin ?5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3? was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3?, and ?-catenin were unchanged. These results suggested that expression of p-GSK-3? was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin ?5.

Dataset Information

Alternagin-C binding to ?2?1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

Publications

Alternagin-C binding to α<sub>2</sub>β<sub>1</sub> integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

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