Cell surface colligin/Hsp47 associates with tetraspanin protein CD9 in epidermoid carcinoma cell lines.

Abstract

Hsps expressed on the cell surface have been associated with tumor invasiveness and used as targets for molecular surveillance. The present study utilized four human oral squamous cell carcinoma cells lines, SCC-4, SCC-9, SCC-15, SCC-25, the murine epidermoid carcinoma cell line LL/2, and primary cultures of human gingival fibroblasts to assess the cell surface expression of colligin/Hsp47, a proposed marker for malignancy. Immunoprecipitation studies following protein crosslinking revealed that Hsp47 was associated with a number of membrane proteins including the tetraspanin CD9. Cytometric analyses were performed to determine the distribution of cell surface colligin/Hsp47 during the phases of the cell cycle. These studies showed that colligin/Hsp47 was not limited to any phase of the cell cycle in epidermoid carcinoma cells. Boyden chamber tumor invasion assays and colloidal gold migration assays utilizing a reconstituted basement membrane (Matrigel), collagen type I, and laminin-5 substrates revealed that cell lines expressing constitutive high levels of colligin/Hsp47 manifested the lowest invasion and migration indices. The incorporation of antibodies against Hsps into the migration and invasion assays, likewise, increased the invasion indices and the phagokinetic migration indices. These data indicate that colligin/Hsp47 is anchored to the cell membrane in a complex with CD9 where it moderates tumor cell invasion and motility possibly by acting as a serpin protein inhibitor or as a receptor for collagen.