Abstract
Purpose: :
We have already shown that the tetraspanin CD9 is involved in migration and adhesion of retinal endothelial cells (iBREC). These cellular processes are deregulated in endothelial cells in diabetic retinopathy (DR). In addition, association of CD9 with metalloproteases, protein kinase C (PKC) and integrins have been described for several cell types and these proteins might play a role in the development of DR. To elucidate the significance of these interactions for retinal diseases, these putative protein complexes were studied in iBREC.
Methods: :
Complex partners of CD9 were identified by co-immunoprecipitation with a CD9-specific antibody followed by Western blotting and/or mass spectrometry using nanoLC-ESI-MS/MS. Their co-localization was verified by double-immunofluorescence staining.
Results: :
Integrin beta-1 (CD29) and integrin alpha-5 (CD49e) which form the classical fibronectin receptor, were identified as complex partners of CD9 in iBREC by double-immunofluorescence staining and/or immunoprecipitation. A portion of the co-localized CD29 and CD49e was also co-localized at the plasma membrane with CD9, whereas intracellular CD9 was not associated with these proteins. In addition the metalloprotease ADAM10 is associated with CD9 as shown by co-immunoprecipitaion. In contrast CD9 did not interact with PKCalpha/beta in iBREC. In addition expression, modification, and cellular localisation of CD9 was not influenced by stimulation or inhibition of PKC.
Conclusions: :
As shown in this study CD9 is associated with the metalloprotease ADAM10 in iBREC. ADAM10 plays an important role in endothelial cell permeability as well as leukocyte recruitment and transmigration. In addition CD9 interacts with those integrin subunits that form the fibronectin receptor. These results are of particular interest in view of new strategies in the treatment of diseases like DR and AMD which also include inhibitors of the fibronectin receptor. Like these, anti-CD9 therapeutics might inhibit choroidal and retinal neovascularization.
Keywords: extracellular matrix • cell adhesions/cell junctions • diabetic retinopathy