Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

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.

• Authors: Alessandra Bosutti, Jie Qi, Roberta Pennucci, David Bolton, Sabine Matou, Kamela Ali, Li-Huei Tsai, Jerzy Krupinski, Eugene B Petcu, Joan Montaner, Raid Al Baradie, Francesca Caccuri, Arnaldo Caruso, Giulio Alessandri, Shant Kumar, Cristina Rodriguez, Jose Martinez-Gonzalez, Mark Slevin
• Journal: PloS one (PLoS One) Vol. 8 Issue 9 Pg. e75538 ( 2013) ISSN: 1932-6203 [Electronic] United States
• PMID: 24098701 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• CDCA5 protein, human
• Cell Cycle Proteins
• DNA Primers
• MEF2 Transcription Factors
• MEF2C protein, human
• Peptides
• Purines
• Roscovitine
• Cyclin-Dependent Kinase 5
• CDK5 protein, human

Topics

• Actin Cytoskeleton (metabolism)
• Adaptor Proteins, Signal Transducing (metabolism)
• Analysis of Variance
• Blotting, Western
• Cell Cycle Proteins (metabolism)
• Cell Line
• Cell Movement (drug effects)
• Colorimetry
• Cyclin-Dependent Kinase 5 (antagonists & inhibitors, genetics, metabolism)
• Cytoskeleton (drug effects)
• DNA Primers (genetics)
• Humans
• Hypoxia (drug therapy, etiology, metabolism)
• Immunoprecipitation
• MEF2 Transcription Factors (metabolism)
• Microarray Analysis
• Microscopy, Fluorescence
• Mutation, Missense (genetics)
• Neovascularization, Physiologic (drug effects, physiology)
• Peptides (metabolism, pharmacology)
• Pseudopodia (metabolism)
• Purines (pharmacology)
• RNA Interference
• Roscovitine
• Signal Transduction (drug effects, physiology)
• Stroke (complications)