The effect of extracellular matrix protein binding and culture confluence status on the effect of ROCK on TNF-α- and IL-1-stimulated CXCL8 secretion by colonic epithelial cell.

Abstract

Colonic and intestinal epithelial cells (EC) attach to a basement membrane of laminins, fibronectin, and collagen IV. Wounding of the epithelial layer can change the types of extracellular matrix (ECM) proteins to which the EC attach. In this study, we determined the effect of culturing Caco-2 cells on different ECM proteins on the capacity of EC to produce TNF-α- or IL-1-stimulated CXCL8. The effect of the ECM proteins was such that CXCL8 secretion by cells cultured on collagen I > collagen IV > fibronectin or laminin-111. However, suppression of ROCK activity resulted in a similar 75 to 85% suppression of CXCL8 secretion regardless of the ECM protein type. This suggests that EC can produce different levels of CXCL8 depending on the type of ECM proteins they attach to, but all cases result in a similar requirement for ROCK activity for optimal CXCL8 secretion. Furthermore, when confluent cells were compared to subconfluent cells, the level of TNF- or IL-1-stimulated CXCL8 secretion was greatly elevated with the subconfluent cells and inhibiting ROCK had no effect on CXCL8 secretion levels by the confluent cells. These experiments suggest that CXCL8 responses by confluent cells, which would model for intact, unwounded epithelial, do not involve ROCK activation. However, CXCL8 responses by subconfluent cells, which would model for cells attaching to and moving on ECM proteins in wounded epithelia, require ROCK activation for greatly elevated CXCL8 responses. These results provide a model to examine the important conditions which regulate chemokine production by EC in wounded epithelia.