A microfabrication approach to study cell-cell interactions in proliferative regulation

Cellular behavior within multicellular organisms is rigorously controlled by numerous cues from the surrounding microenvironment. In particular, adhesive interactions with the ECM and with neighboring cells together coordinate the regulation of cell proliferation. Using microfabrication approaches to engineer cellular microenvironments, we examined the role of cell-cell interactions in proliferative signaling.; We first investigated whether cell-cell adhesion is involved in transducing mechanical stretch into a proliferative response in vascular cells. Using microcontact printing to pattern cells on flexible membranes, we isolated the roles of cell-cell and cell-matrix adhesions and demonstrated that endothelial cells, but not smooth muscle cells, required cell-cell contact and VE-cadherin to transduce stretch. Furthermore, endothelial cells required Rac1 while smooth muscle cells required RhoA to proliferate in response to stretch. These data suggest that cadherins are important for mechanotransduction, and that endothelial and smooth muscle cells use distinct mechanisms to respond to stretch.; We then examined the role of E-cadherin engagement in epithelial cell proliferation. We observed that epithelial cells exhibited a biphasic proliferative response to seeding density. Experiments using microwell substrates showed that cell-cell contact mediated by E-cadherin engagement stimulated proliferation, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading. Stimulation of proliferation by E-cadherin involved Rac1 activity and p120-catenin. These findings demonstrate a positive role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell-cell contact may trigger or inhibit proliferation in different settings.; Finally, we developed a method to examine contact-mediated proliferation in three-dimensional cultures. We formed structures of different sizes and configurations by releasing cells from two-dimensional micropatterns. Analogous with observations of cells on two-dimensional surfaces, peak levels of proliferation were found in cells within intermediate-sized clusters. These experiments illustrate a technique to control the spatial organization of cells in three-dimensional cultures, and suggest that cell-cell contact also stimulates proliferation in a three-dimensional setting.; Together, these studies demonstrate a stimulatory role for cell-cell adhesion, and cadherins in particular, in regulating proliferation, and highlight the value of microengineered tools for understanding the role of cell-cell adhesion in cell biology.