Activation of protein kinase C by phorbol esters modulates α2β1 integrin on MCF-7 breast cancer cells

Abstract

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell- substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-1,3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4- h interval. This enhanced adhesion was mediated by α2β1 integrin, since both anti-α2 and anti-β1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of α2β1 integrin over a 4-h time interval. However, α2β1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of α2β1-dependent cellular adhesion preceded the induction of α2β1 cell surface expression. Northern blot analysis revealed that mRNA levels of both α2 and β1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of α2β1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of α2β1 was independent of increased expression of α2β1. Protreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of α2β1 integrin expression and of α2β1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of α2β1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3, C3 inhibited TPA- induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the α2β1-dependent adhesion of MCF- 7 cells by two distinct mechanisms: altering the gene expression of integrins α2 and β1 and altering the avidity of the α2β1 integrin by a Rho- dependant mechanism.