Roles of focal adhesions and fibronectin-mediated cohesion in proliferation of confluent fibroblasts.

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density-dependent growth-arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor-beta1, ascorbic acid, and serum. This proliferation depended on alpha 5 beta 1-integrin-mediated cell-FN-cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell-FN-cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Density-arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin-, paxillin-, and phosphotyrosine-containing FAs throughout the ventral cell-surface, indicating ROCK-mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y-27632 or the myosin II inhibitor blebbistatin, the up-regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of alpha 5 beta 1-integrin-mediated cell-FN-cell cohesion. The present findings provide insights into the rational design of high-density fibroblast transplants.