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Payam Falatoonzadeh, Grace Woo, Nitin Udar, Shari Atilano, Marilyn Chwa, Miceli Michael, S Michal Jazwinski, Cristina Kenney; An In-Vitro Study of Wnt Pathway Modulation in Cybrid H and J Mitochondrial Haplogroups and the Possible Implications for Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4998.
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The wet form of age-related macular degeneration (AMD) can be associated with increased VEGF, vascular cell proliferation, and induced choroidal neovascularization. Mitochondrial DNA (mtDNA) haplogroups are defined by accumulation of SNPs that represent different geographic origins of populations. Individuals with haplogroup H mtDNA are lower risk for AMD, while haplogroup J individuals have higher predisposition. The Wnt pathway and β-Catenin, one of its downstream proteins, are commonly associated with cell proliferation, and studies have focused on its role in angiogenesis related to cancer and possibly AMD. Our present study uses a mitochondrial haplogroup cytoplasmic hybrid (cybrid) model to assess the effects of mtDNA variations on the Wnt pathway.
Mitochondria-deficient (Rho0) human ARPE-19 cells were fused with platelets from individuals with mtDNA haplogroups H (n=3) or J (n=3) to establish cybrid cell lines. RNA was extracted from each cybrid and cDNA synthesized. Q-PCR was performed using primers for various genes associated with the Wnt pathway and analyzed for changes in gene expression levels of J cybrids relative to H cybrids.
Gene expression levels were significantly decreased in J cybrids relative to H cybrids for inhibitors of the Wnt pathway, including SFRP1 (0.29 fold, p=0.0001), DKK3 (0.46 fold, p=0.004), and RARA1 (0.57 fold, p=0.007). There was also a relative increase in expression of GSK3 (1.6 fold, p=0.005). Expression levels of other members of the Wnt pathway, including CSNK1E, WNT9A, LRP1, KREMEN1, MDM2, and TGFβ were not found to be significantly different in the J versus the H cybrids.
SFRP1 and DKK3 inhibit the binding and interaction of the Wnt ligand with cell surface receptors. Decreased expression of these genes in J cybrids could potentially lead to more Wnt ligand binding and increased signal transduction within the cell. This would include recruitment of increased GSK3A away from the β-catenin degradation complex. Also, decreased RARA1 levels, a nuclear inhibitor of β-catenin, would allow for more β-catenin driven transcription. These observations correlate with faster cybrid J growth patterns in culture, and may also have implications for how different haplogroups might modulate the Wnt pathway and possibly influence AMD.
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