Abstract
Purpose: :
Caveolins belong to a family of scaffolding membrane proteins which are the principal components of caveolae membranes. Recently a genome-wide association study for POAG patients identified a new common glaucoma-associated sequence variant at chromosome 7q31 (rs4236601 A) located close to caveolin genes (CAV1 and CAV2). These genes are expressed in the trabecular meshwork (TM) and retinal ganglion cells, two types of cells implicated in the pathogenesis of POAG. We investigated expression patterns and regulation of caveolins in TM primary cultures and immortalized cells.
Methods: :
We used two immortalized cell lines derived from normal (NTM-5) and glaucomatous (GTM-3) TM (generous gift from Alcon Research Ltd, Fort Worth, TX). Extracts from these cells were separated in 10% PAAG, Western blotted and tested with CAV1 and CAV2 Abs, followed by scanning densitometry. To study a possible effect of synucleins - lipid binding proteins expressed in TM and retinal ganglion cells (RGC)- on CAV expression the cells were transiently transfected with vector expressing three members of the synuclein family and the level of CAV1 was determined by Western blot (WB).
Results: :
We found that CAV1 and CAV2 are expressed in both NTM-5 and GTM-3 as monomers with mol.weight ~21-24 KDa. The expression level of CAV2 in GTM-3 is 2.1 times lower than in NTM-5. After 24 h of growth, up to 70% of CAV1 is presented as aggregates with mol. weight 250 KDa, however, aggregates disappear after 48 h of cell growth. After cell treatment with dexamethasone CAV1 expression is upregulated 2.4 times, whereas CAV2 expression remains unaltered. CAV1 and CAV2 have a similar cytoplasmic/ membrane intracellular localization in both types of cultures. Synuclein family members differentially affect CAV1 expression with the strongest effect of alpha-synuclein (two-fold upregulation in NTM-5 cells). After immunoprecipitation with polyclonal CAV-1 specific Ab, the presence of both monomeric CAV1 and CAV1 aggregates was revealed with monoclonal Ab. Tyr14 in aggregated CAV1 was not phosphorylated.
Conclusions: :
Monomeric CAV1 and its aggregates are in dynamic equilibrium in immortalized TM cells. Alpha-Synuclein upregulates CAV1 expression in TM cells in a similar way as in the brain neuronal cultures. Preliminary data indicate that CAV1 and synucleins are colocalized in TM cells and cooperate in the regulation of signaling pathways. The knowledge of the regulation of caveolins expression and their role in intracellular signaling in TM is important for better understanding of glaucoma pathogenesis.
Keywords: trabecular meshwork • gene/expression • cell membrane/membrane specializations