Abstract
Many studies have shown that growth factors and cell adhesion both regulate cell cycle progression through G1 phase, and that receptor tyrosine kinase and integrin signaling pathways are interconnected. My main hypotheses were that (i) RTK and integrin signaling cooperate in regulating cyclin D1 expression, and that (ii) a component of this integrin signal is mediated through the actin cytoskeleton. To facilitate these studies, NIH-3T3 fibroblasts stably expressing human alpha5 integrin (halpha5-3T3 fibroblasts) were attached to fibronectin or anti-human alpha5beta1 and treated with growth factors. I found that cooperative signaling between growth factor receptors and alpha5beta1 integrin was required for sustained ERK signaling. This effect explained, at least in part, the combined growth factor and anchorage requirements for the expression of cyclin D1 in mid-G1 phase. Inhibition of Rho or Rho kinase disrupted stress fibers, blocked sustained ERK signaling and, consequently, inhibited mid-G1 cyclin D1 expression. It also revealed a strikingly premature (as early as 1 h in G1 phase) induction of cyclin D1. Studies with constitutively active and dominant negative mutants showed that a rac/cdc42 pathway regulates this early G1 phase expression of cyclin D1, in an ERK- and stress fiber-independent manner. Thus, Rho plays a unique role in determining the timing of cyclin D1 expression within G1 phase: permitting the coupling of sustained ERK activation and midG1 phase expression of cyclin D1, while suppressing an alternate rac/cdc42-dependent pathway that results in early G1 induction of cyclin D1. Analysis of Rho kinase effector pathways demonstrated that sustained ERK activity required Rho kinase-dependent stress fiber formation and integrin clustering, while suppression of the rac/cdc42 pathway was regulated by LIM kinase. Moreover, I showed that LIM kinase is acting through a novel mechanism, independent of its stimulatory effect on stress fiber formation. Overall, my research has (i) identified two distinct pathways by which growth factors and extracellular matrix regulate the expression of cyclin D1, (ii) demonstrated that the relative use of these pathways results in distinct patterns of cyclin D1 expression, and (iii) emphasized that Rho acts as a switch that regulates the activity of these pathways and, thereby, controls the duration of G1 phase.