Depletion of αV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis

Integrin heterodimers sharing the common alpha V subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alpha V integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alpha V beta 3 and alpha V beta 5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alpha V integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor aV integrin subunit was not affected. However, the maturation of alpha V integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of alpha V integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, alpha V integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation. (C) 1999 Elsevier Science B.V./International Society of Matrix Biology. All rights reserved.