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Expression of caveolin in trabecular meshwork cells and its possible implication in pathogenesis of primary open angle glaucoma

2011-11-03 22:05:15

Molecular Vision; 2011 Nov; 17:2878-88


Irina Surgucheva and Andrei Surguchov



Abstract



Purpose:



Primary open-angle glaucoma (POAG), which is the most common form of glaucoma, has been associated with a heterogeneous genetic component. A genome-wide association study has identified a common sequence variant at 7q31 (rs4236601 [A]) near the caveolin genes in patients with POAG. Caveolins are a family of integral membrane proteins which participate in many cellular processes, including vesicular transport, cholesterol homeostasis, signal transduction, cell adhesion and migration. The goal of this study was to investigate the expression and regulation of caveolin 1 (CAV-1) and caveolin 2 (CAV-2) in normal and glaucoma trabecular meshwork (TM) cells.



Methods:



CAV-1 and CAV-2 protein expression was quantified by immunoblot analysis using lysates isolated from primary and immortalized TM cells or TM tissue dissected from normal and POAG eyes. The localization of caveolins in TM cells was assessed by immunofluorescent microscopy. CAV-1 and CAV-2 protein expression was also investigated in TM cells at various time points after subjecting the cells to known glaucomatous insults like dexamethasone (DEX) and tumor growth factor beta2 (TGF-β2) treatment. Phosphorylation of CAV-1 at tyrosine 14 in normal and glaucoma TM cell lines was evaluated using a specific monoclonal antibody (Ab). The 5' upstream region of the CAV-1 gene was amplified and the sequence variant rs4236601 (A/G polymorphic site) and several putative transcription factor-binding sites were modified by in vitro mutagenesis. The effect of nucleotide sequence modifications in the CAV-1 upstream region on gene expression was assayed in a luciferase-based system in TM and non-TM cells.



Results:



CAV-1 and CAV-2 are expressed in TM cells, with localization to the cytoplasm and perinuclear region. DEX increased CAV-1 expression in immortalized glaucoma TM cells by 2.8±0.1 (n=3) fold at 24 h and 2.5±0.1 (n=3) fold at 48 h, compared to 1.3±0.06 (n=3) fold at 24 and 48 h in immortalized normal TM cells. Phosphorylation of CAV-1 at Tyr14 was reduced by 3.2±0.15 (n=3) fold in glaucomatous TM cells when compared to normal TM cells. In POAG and normal TM tissue, CAV-1 expression was found to be uniform. CAV-2, on the other hand, was variable in independent normal and glaucoma TM tissue. Substitution of a G for an A at base pair -2,388 upstream of the start codon of CAV-1, corresponding to the minor allele rs4236601 [A], increased transcriptional activity in TM and non-TM cells when compared to the native sequence. Deletion analysis of putative transcription factor binding sites in the CAV-1 promoter region caused cell-specific effects on gene expression.



Conclusions:



CAV-1 and CAV-2 are expressed in normal and glaucoma tissue and TM cell lines. Phosphorylation of Tyr14 in CAV-1 and transcriptional regulation of CAV-1 expression may have a role in glaucomatous alterations in TM cells.



Introduction



Primary open angle glaucoma (POAG), which is the most common form of glaucoma, is the second leading cause of blindness worldwide, affecting nearly 70 million people. POAG usually affects older people and, with the aging of world populations, it is predicted that the number of patients with the disease will substantially increase over the next decade. The prevalence of POAG among Americans aged 40 and older is estimated at 1.86 percent, affecting 2.22 million individuals.



The disease mainly affects the optic nerve and retinal ganglion cells leading to visual impairment and blindness. Of the various risk factors of POAG, elevated intraocular pressure (IOP) is the most prevalent and clinically relevant. The elevation of IOP results from impaired drainage of aqueous humor through the conventional pathway, which consists of the trabecular meshwork (TM), Schlemm’s canal (SC), collector channels and aqueous veins.



Recently, a genome-wide association study identified the first common genetic risk factor for POAG, corresponding to a region of the genome on chromosome 7q31. The locus spans the caveolin 1 (CAV-1) and caveolin 2 (CAV-2) genes and the minor allele of a genetic marker, rs4236601, located in the noncoding upstream region of CAV-1, was found to be tightly associated with POAG. However, a smaller population based study suggests that the rs4236601 polymorphic allele may not be a universal risk factor in all populations. Although the association does not mean that caveolins are directly implicated in glaucoma, the finding has prompted interest in caveolin family members and their role in cells of the conventional outflow pathway.



Caveolins are the signature proteins of caveolae, flask-shaped invaginations of plasma membrane that are 50–100 nanometers in diameter. Caveolins are scaffolding proteins with molecular weight 22–24 kDa embedded in the cytosolic leaflet of cell membranes, with both NH2- and COOH-termini residing in the cytosol. Caveolins interact with and compartmentalize membrane-localized signaling proteins to facilitate and modulate high-fidelity intracellular signaling pathways. In addition, they participate in many important cellular processes such as vesicular transport (including cell to cell protein and microRNA delivery), adhesion and cell motility, cholesterol homeostasis and tumor suppression. Caveolin structure, function and role in human diseases have been extensively investigated in different tissues and organs.



There are three caveolin proteins: CAV-1, CAV-2, and caveolin 3 (CAV-3) which form a structural backbone of caveolae. CAV-1 and CAV-2 have overlapping patterns of expression throughout numerous tissues, whereas CAV3 has more restricted expression in muscle and in the nervous system. CAV-1 and CAV-2 genes are located within 19 kilobases on human chromosome 7q31.1 (Figure 1), while CAV-3 is mapped to chromosome 3p25. Several cell-type specific transcription factor (TF) binding sites ensuring cell and tissue-specific expression have been described in the caveolin promoter region. Due to the reported genetic association of a marker near CAV-1 and CAV-2 with POAG, we analyzed their expression and regulation in normal and glaucomatous TM cells. Additionally, we evaluated the role of cis-elements upstream region of CAV-1 and the effect of the recently described G-A polymorphism located in the upstream region of CAV-1 (Figure 1) in transcriptional regulation.



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