A Switch between ERK and Rac signaling regulates cyclin D1 expression and G1 phase progression of quiescent and cycling cells

Jessie Villanueva, Kristin Roovers, and Richard K. Assoian
Department of Pharmacology, University of Pennsylvania School of Medicine,
167 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia PA 119104-6084, USA

INTRODUCTION.  The rate of cell cycle progression through G1 phase is, in large part, determined by the induction of cyclin D1, typically the rate-limiting step in formation of cyclin D-cdk4/6 complexes and the consequent sequestration of cdk inhibitors that regulate cyclin E-cdk2. Once active, cdk4/6 and/or cdk2 phosphorylate the retinoblastoma protein. Allowing for the release of free E2Fs and the induction of E2F-dependent genes, such as cyclin A, required for S phase entry and cell cycle progression.

ERK (p42/p44 MAPK) constitutes a major signaling pathway that is required for G1-phase cell cycle progression after mitogenic stimulation of quiescent cells. ERK mediates the induction of cyclin D1 in mid-G1 phase, and a sustained ERK signal (typically ~6 h after the release from quiescence) is thought to be required for this effect ^{(1, 2)}.   We have analyzed the effect of MEK/ERK signaling on the G1 phase cyclins and cyclin-dependent kinase inhibitors to identify essential ERK targets during G1 phase progression. 

METHODS. Spontaneously established MEFs, early passage human foreskin fibroblasts and early passage murine aortic smooth muscle cells were cultured and rendered quiescent by serum-starvation as previously described ^(2,3,4). To maintain continuously cycling cells, cultures were incubated in DMEM with 10% FBS, kept at or below 50% confluence and fed fresh medium containing 10% FBS twice 1 day before the experiment. Transient transfections were performed as described⁽²⁾. In some experiments, the cells were incubated with 50 mM U0126 (Promega) or DMSO (vehicle).  U0126 was washed-out by rinsing monolayers twice with cold DMEM and then re-feeding the cultures fresh DMEM-10% FBS.  MEFs expressing a tetracycline-repressable cyclin D1⁽⁵⁾ were cultured similarly except that they were maintained in the presence of 2 mg/ml tetracycline and then serum-starved and stimulated in the absence and presence or 2 mg/ml tetracycline. S phase entry was determined by performing the serum stimulations in the presence of 3 mg/ml BrdU and then determining the incorporation of BrdU in cells counterstained with Dapi (Roovers and Assoian, 2003). Northern blotting of total RNA used random-primed cDNA probes and standard procedures. Western blotting was performed as described ⁽⁵⁾_.

RESULTS. By sequentially adding and removing the MEK inhibitor U0126, we found that early G1 phase ERK activity is dispensable for the induction of cyclin D1 and entry into S phase in mouse and human fibroblasts.  In fact, we find that if cyclin D1 is expressed ectopically, at near endogenous levels, or through an endogenous Rac/Cdc42 pathway⁽²⁾, ERK activity becomes dispensable for all of G1 phase, indicating that cyclin D1 is the sole essential ERK target during the first G1 phase in fibroblasts.  We then examined the role of ERK signaling for cyclin D1 expression and S phase entry in continuously cycling cells. Surprisingly, we found that ERK activity was dispensable for both cyclin D1 induction and S phase entry. Furthermore we show that cyclin D1 induction is mediated by the Rac/Cdc42 pathway in these conditions and no other cell cycle event required Rac/Cdc42 activity. Our results show that ERK activity plays a limited role in G1 phase control and that a switch from ERK to Rac/Cdc42 regulates cyclin D1 expression as cells progress from the first to subsequent G1 phases. 

DISCUSSION. Many studies exploring the relationship between ERK signaling and G1 phase cell cycle progression have focused on relatively short-term effects after mitogenic stimulation of quiescent fibroblastic cells.  Our results emphasize that these short-term ERK effects will not be required for G1 phase progression if they do not persist into mid-G1 phase.  Moreover, our data indicate that even sustained ERK activity is only required for progression through the first G1 phase (as quiescent cells re-enter the cycle) and that the principal signaling system regulating cyclin D1 production during subsequent cell cycling is Rac/Cdc42, at least for fibroblastic cells.  Our results reveal a very limited role for ERK1/2 in G1 phase progression and describe a fundamental difference in the signal transduction cascades regulating cyclin D1 expression and G1 phase progression in quiescent and cycling cells.   Short-lived ERK signals are dispensable for G1 phase progression.  Sustained ERK signaling is required for both cyclin D1 expression and G1 phase progression upon cell cycle re-entry from quiescence, but this requirement is observed only because Rac signaling to cyclin D1 is repressed under that condition.  The dispensability of ERK signaling in cycling cells, and in quiescent cells expressing activated Rac or cyclin D1, has potential implications for the therapeutic use of MEK inhibitors as antagonists of G1 phase progression.

REFERENCES 

1. Roovers, K., G. Davey, et al. (1999). "Alpha5beta1 integrin controls cyclin D1expression by sustaining mitogen-activated protein kinase activity in growth factor-treated cells." Mol Biol Cell 10(10): 3197-204.
2. Welsh CF, Roovers. K., Villanueva J, Liu Y, Schwarts MA, Assoian RK (2001). "Timing of cyclin D1 expression within G1 is controlled by Rho." Nat Cell Biol 3: 950-957.
3. Bohmer, R. M., E. Scharf, et al. (1996). "Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1." Mol Biol Cell 7(1): 101-111.
4. Kothapalli, D., Stewart, S.A., Smyth, E.M., Azonobi, I., Rader, D.J., Pure, E., and Assoian, R.K. (2003) IP activation inhibits proliferation of aortic smooth muscle cells by regulating CREB- and pocket protein-dependent cyclin A gene expression, submitted.
5. Roovers, K. and R. K. Assoian (2003). "Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases." Mol Cell Biol 23(12): 4283-94.