Between ROCK and a Hard Place

Abstract

Mammary epithelial cells grown in three-dimensional (3D) floating collagen gels differentiate to form ductlike tubular structures, whereas cells grown in a 3D collagen matrix attached to the bottom of a culture dish do not. Wozniak et al. found that mammary epithelial cell lines grown in floating gels, which formed tubules and contracted the gels, showed increased staining for Ki67 (a marker of proliferation) compared with cells grown in attached matrices (which could not contract gels and did not form tubules). Inhibition of the small guanosine triphosphatase Rho or its effector ROCK (Rho-associated kinase) blocked gel contraction and tubule formation, as did inhibition of actin-myosin contractility (downstream of ROCK) or expressing constitutively active or dominant-negative Rho mutants, or constitutively active Rho guanine nucleotide exchange factors. Western analysis indicated that Rho activity was down-regulated in cells grown in floating gels compared with cells grown without collagen or attached gel. Cells transfected with a kinase-dead ROCK mutant or grown in the presence of ROCK or actin-myosin inhibitors maintained high Rho activity in floating gels, which suggests ROCK-dependent feedback inhibition of Rho and the need for precise regulation of the ROCK pathway. Growing cells in floating gels made with high concentrations of collagen (which interfered with contraction) also maintained high Rho activity and inhibited tubule formation. Because dense breast tissue (characterized by increased deposition of collagen) is associated with an increased risk of breast cancer, this research may have pathophysiological implications. M. A. Wozniak, R. Desai, P. A. Solski, C. J. Der, P. J. Keely, ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J. Cell Biol . 163 , 583-595 (2003). [Abstract] [Full Text]