- Authors: Matou-Nasri, S., Gaffney, J., Kumar, S., Slevin, M.
- Year: 2009
- Journal: Int J Oncol 35 761-73
- Applications: in vitro / Protein/Peptide/Antibody / PULSin
- Cell type: BAEC
Description: Bovine arterial endothelial cells
We used short-interfering RNA (siRNA) to knockdown the hyaluronan (HA) receptors CD44 and the receptor for hyaluronan-mediated motility (RHAMM) in vascular endothelial cells to investigate their role in angiogenesis. We showed that CD44 and RHAMM single knockdown inhibited low molecular weight hyaluronan (o-HA)-induced endothelial cell tube formation in Matrigel, but no change in the control, epidermal growth factor-induced tube formation was observed. Using a Kinexus phosphoprotein array and confirmational Western blotting we were able to show a differential effect on HA-induced protein expression after CD44 and RHAMM knockdown. CD44 knockdown abolished o-HA-induced membrane phospho-protein kinase C-alpha (PKC-alpha) and down-stream phospho-gamma-adducin expression. Using the PKC inhibitor Go6976, we demonstrated the involvement of PKC-alpha and gamma-adducin in o-HA-induced tube formation, whilst o-HA-induced enzymatic activity of MMP9 was also reduced. This suggests that endothelial tube formation involves activation of MMP9 via PKC-alpha. Furthermore, the involvement of gamma-adducin in o-HA-induced F-actin cytoskeleton rearrangement was CD44-dependent and the reduction of CD44 expression lead to a change in endothelial cell morphology. Both RHAMM and CD44 knockdown completely inhibited o-HA-induced Cdc2 (Cdk1) phosphorylation suggesting a possible involvement in cell cycle control. Although CD44 and RHAMM are both involved in o-HA-induced endothelial tube formation in Matrigel, they mediate distinct angiogenic signalling pathway and for the first time we demonstrated the specific involvement of gamma-adducin in CD44/o-HA-induced endothelial tube formation. The data presented here extend our understanding of key stages of the processes of o-HA-induced angiogenesis which may have relevance to tumour progression.