Differential Activation of ERK and Rac Mediates the Proliferative and Anti-proliferative Effects of Hyaluronan and CD44

Devashish Kothapalli,James Flowers,Tina Xu,Ellen Puré,Richard K Assoian

doi:10.1074/jbc.m802934200

Abstract

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Highlights

LMW-HA Synergizes with a Suboptimal Mitogenic Stimulus to Promote S Phase Entry—We previously reported that LMW-HA stimulates S phase entry in quiescent mouse vascular smooth muscle cells (SMCs) incubated in suboptimal (Ͻ1%) FBS, and that the effect requires CD44 [23]
We examined the effect of that the increased inactivation of Rb and S phase entry seen in these pathways on FBS-stimulated cyclin D1 gene expression in response to LMW-HA results from up-regulation of cyclin D1, human vascular SMCs and asked if these pathways might be down-regulation of p27, and the consequent activation of con- differentially regulated by HMW-HA and LMW-HA
The results described here document the mechanism underlying the pro-mitogenic effect of LMW-HA, and begin to explain how HMW-HA and LMW-HA can have opposing effects on cell cycle progression despite binding to the same receptor, CD44

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Early passage human vascular SMCs were maintained and serum-starved at near confluence for 48 h as described [20]. The solutions were supplemented with FBS (to 10 or 0.1% final concentrations for experiments with HMW-HA or LMW-HA, respectively) and directly added to the serum-starved cells. The infected, starved cells were washed once with serum-free DMEM before being incubated with 0.1% FBS in the presence or absence of LMW-HA (200 ␮g/ml). For siRNA-mediated knockdown, human vascular SMCs were trypsinized and reseeded (106 cells per 100-mm dish) in DMEM, 10% FBS without antibiotic overnight. The day, Lipofectamine 2000 (Invitrogen; 1 ␮l per 25,000 cells) and siRNAs (Ambion; 150 nM final concentration) were diluted in Opti-MEM (Invitrogen) and added to the confluent cells for 4 h before serum-starving the cells for an additional 24 h in fresh Opti-MEM [30]. Rac Activation Assay—Near-confluent SMCs (ϳ105 cells per 35-mm dish) were serum-starved for 48 h

The cells were preincubated with

Quantitative Real Time Reverse

RESULTS

DISCUSSION