Restricted Expression and Function of Laminin 1-Binding Integrins in Normal and Malignant Oral Mucosal Keratinocytes

Abstract

Squamous cell carcinoma of the oral cavity spreads by initial invasion of the laminin-rich basement membrane. We examined the adhesion and motility of human oral SCC cells and normal mucosal keratinocytes and found that the SCC cells readily attached and migrated on laminin 1 substrates but migrated poorly on collagen type I and fibronectin. The normal keratinocytes, however, adhered poorly to and were non-motile on laminin 1 yet readily and preferentially attached and migrated on fibronectin and collagen type I. Analysis with blocking anti-integrin antibodies showed that the SCC cells used the α6β1 complex to attach and migrate on laminin 1 and that this activity was confined to the E8 long arm fragment of laminin. Affinity chromatography on laminin-Sepharose columns revealed that the SCC cells, but not normal keratinocytes, expressed high levels of the α6β1 laminin 1 receptor. Metabolic pulse-chase analysis indicated that in contrast to the SCC cells, keratinocytes did not have a stable pool of β1 subunit precursor. Preferential pairing of α6 with β4 and the deficiency in pre-β1 levels appear to account for the failure of keratinocytes to form significant α6β1 complex. Additionally, the presence of laminin 1 in co-coating experiments blocked keratinocyte adhesion to other immobilized ligands, such as collagen type I or fibronectin. This anti-adhesive effect seemed to reflect a general paralysis of cell adhesive function, since laminin 1 also diminished the adhesion of keratinocytes to substrates coated with immobilized anti-integrin subunit antibody. The inhibitory activity of laminin 1 resided in the E1' and E8 fragments, and not in the E3, E4 or G domains. Collectively, our results indicate that laminin 1 is a restrictive ligand for normal keratinocytes, apparently because of their failure to assemble and express the α6β1 complex or other functional laminin receptors and their sensitivity to the anti-adhesive activity of laminin itself. The elevated expression of α6β1 following malignant conversion of mucosal keratinocytes promotes their migration on laminin, a process important during invasion and metastasis.