Oligosaccharides of hyaluronan induce angiogenesis through distinct CD44 and RHAMM-mediated signalling pathways involving Cdc2 and gamma-adducin.
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.
Matou-Nasri
S
S
School of Biology, Chemistry and Health Science, Manchester Metropolitan University, Manchester M1 5GD, UK.
Gaffney
J
J
Kumar
S
S
Slevin
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D000099291:Adducins
D000199:Actins; D016203:CDC2 Protein Kinase; D002148:Calmodulin-Binding Proteins; D002227:Carbazoles; D004815:Epidermal Growth Factor; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D020780:Matrix Metalloproteinase 9; D009844:Oligosaccharides; D051571:Protein Kinase C-alpha; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D000099291:Adducins; C098946:hyaluronan-mediated motility receptor; C081021:Go 6976