Abstract 243 Poster Session III, Monday, 5/3 (poster 101)

Estrogen appears to play a significant role in the regulation of vascular cell function. Estradiol (E2) augments endothelial cell (EC) activities related to vascular healing and angiogenesis, enhancing EC attachment, migration, proliferation and differentiation in vitro. Previously, our laboratory defined a basic fibroblast growth factor (bFGF)-mediated autocrine loop by which bFGF stimulates ERK MAP kinase activation in EC. Here, we report initial characterization of E2 effects on the cell cycle machinery. Association of cell cycle-regulatory cyclins with cyclin-dependent kinases (CDKs) and induction of CDK activity are important events in cell cycle transitions. Cell cycle progression is inhibited by association of the cyclin kinase inhibitors (CKIs), p21 and p27, with the cyclin/CDK complex.

To define the role of E2 in these events, actively proliferating human umbilical vein EC were E2 depleted by culture in hormone-free, phenol red-free media for 24 hours. Cells were then treated with E2 (3 nM) for 3h, 6h, or 24h. Cyclin-associated CDK activity was evaluated by an in vitro kinase assay. E2 depletion resulted in a decrease in both cyclin D1- and cyclin E-associated kinase activity. E2 treatment induced cyclin D1 and cyclin E associated kinase activity as early as 3h, with maximal activation after 6h of treatment and sustained activity through 24h. Induction of CDK4 activity followed the same time course as that for cyclin D1-associated kinase activation. These events occurred without changes in protein levels for cyclins D1 or E, CDK2, or CDK4, indicating that stimulation reflected increased activation rather than increased protein expression. Protein levels of the CKIs, p21 and p27, did not change with E2 depletion or treatment. Cyclin association with CDKs and CKIs was then evaluated by immunoprecipitation and western blot. E2 treatment decreased association of p21, but not p27, with immunoprecipitated cyclin D1.

In summary, E2 induces cyclin-associated CDK activity in E2-depleted EC in vitro. In contrast to other systems, activation does not seem to depend on an increase in cyclin or CDK protein, or a decrease in CKI protein. Rather, the kinase activation may result from E2-induced dissociation of the CKI from the cyclin-CDK complex. As we have previously shown that E2 augments bFGF-stimulated ERK signaling, E2 could induce CDK activation via enhancement of growth factor-stimulated cell cycle induction. These data suggest that E2 effects on vascular EC may include induction of cell cycle regulatory components.