Abstract
Progression of eukaryotic cells through the cell cycle requires the activation of many different intracellular signal transduction pathways. These signals are generally initiated by growth factors which cooperate with integrins to promote activation of the appropriate signaling cascades. In many cases Ras plays a central role in both cell cycle progression and transformation through the activation of different downstream effectors, such as the MEK/ERK pathway, PI3K pathway and signaling molecules associated with the small RHO GTPases. These three signaling pathways have the ability to regulate cyclin D1 expression at different levels and therefore promote G1 phase cell cycle progression. I have studied the role of these signaling pathways for cyclin D1 gene expression, as fibroblasts progress through G1 into the S phase of the cell cycle. I have found that adhesion to the ECM regulates cyclin D1 expression at least in part by sustaining ERK activation in MEFs. In addition, adhesion to the ECM allows for subsequent cell spreading, Rho-mediated stress fibers formation and cytoskeletal organization. All these events are required to sustained ERK activation and to allow for mid-G1 cyclin D1 gene expression. Moreover, forced expression of a constitutively active form of MEK rescues sustained ERK activation in the absence of adhesion or Rho-mediated actin-cytoskeletal organization. I have determined also the exact timing when the ERK signaling cascade needs to be active to induce mid-G1 phase cyclin D1. I found that ERK activity is only required for cyclin D1 gene expression in mid-G1, between 3--6 hours after the release of quiescence, while early G1 ERK activity is completely dispensable for both cyclin D1 gene expression and S phase entry. Similarly, PI3K is only required in mid-late G1 for cyclin D1 gene expression and S phase entry. More importantly, while, these two pathways collaborate to allow for cyclin D1 gene expression in mid-G1 phase, the major ERK target in G1 phase is cyclin D1, while PI3K plays additional roles in cell cycle progression. Interestingly, cyclin D1 gene expression is regulated by ERK only as cells reenter G1 phase from quiescence; cyclin D1 expression becomes ERK-independent in cells that are continuously cycling. Finally, using nuclear run-on assays, I have determined that adhesion to the ECM regulates cyclin D1 at the transcriptional level and that cytoskeletal organization, ERK and PI3K signaling are required to activate the cyclin D1 promoter.