Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta1 integrin.
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.
Gama-de-Souza
Letícia N
LN
Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Cyreno-Oliveira
Elaine
E
Freitas
Vanessa M
VM
Melo
Edielle S
ES
Vilas-Boas
Vanessa F
VF
Moriscot
Anselmo S
AS
Jaeger
Ruy G
RG
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D003528:Carcinoma, Adenoid Cystic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism / Q000494:pharmacology*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D009208:Myoepithelioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012468:Salivary Gland Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000502:physiology; D053668:Syndecan-1 / Q000235:genetics / Q000378:metabolism*
C105786:AG 73; D002614:Chelating Agents; D004151:Dipeptides; D019012:Integrin beta1; D007797:Laminin; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010446:Peptide Fragments; D034741:RNA, Small Interfering; C508241:SDC1 protein, human; D053668:Syndecan-1; C084098:laminin A; D006493:Heparin; D020782:Matrix Metalloproteinases; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D002118:Calcium