RESUMO
We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.
Assuntos
Integrina beta1/metabolismo , Laminina/farmacologia , Metaloproteinases da Matriz/metabolismo , Fragmentos de Peptídeos/farmacologia , Neoplasias das Glândulas Salivares/patologia , Sindecana-1/metabolismo , Western Blotting , Cálcio/metabolismo , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quelantes/farmacologia , Dipeptídeos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Heparina/farmacologia , Humanos , Imuno-Histoquímica , Integrina beta1/genética , Laminina/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/genética , Mioepitelioma/genética , Mioepitelioma/metabolismo , Mioepitelioma/patologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo , Transdução de Sinais/fisiologia , Sindecana-1/genéticaRESUMO
Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin alpha1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of alpha3 and alpha6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that alpha3, alpha6, and beta1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through alpha3beta1 and alpha6beta1 integrins and the ERK 1/2 signaling pathway.