Abstract
Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.
Highlights
The importance of angiogenesis in relation to neuronal replenishment and survival after stroke has been clearly demonstrated
Cyclin-dependent kinase-5 (Cdk5) and p35/p25 are concomitantly overexpressed in endothelial cell (EC) in hypoxic regions of stroke tissue [10], suggesting that the balance of signaling between these pathways may help to define cellular fate in relation to angiogenesis or cell protection after stroke [13]
Our study demonstrates that Cdk5 activation through its major substrate p35 provides a key trigger for initiation and maintenance of in vitro brain EC angiogenesis and that this interaction and signalling cascade is crucial for ensuring correct cytoskeletal organisation/dynamics, involving integrin beta-1 and talin, and allowing cell spreading and subsequent cell migration
Summary
The importance of angiogenesis in relation to neuronal replenishment and survival after stroke has been clearly demonstrated. In this respect, revascularization and associated reperfusion are vital determinants of tissue survival and patient recovery after stroke and a major potential target for successful therapies [1]. Angiogenesis “per se” is a tightly regulated multi-step process, by which new blood vessels are formed from endothelial cell (EC) sprouts, emanating from preexisting vessels and is mediated by the combination of a complex range of angiogenic and anti-angiogenic factors, driving the recruitment, migration, proliferation and differentiation of ECs [2]. The Cdk is emerging as a potential important regulator of vasculogenesis [5]
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