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Sarah Melissa Porras Jacobo, Andrius Kazlauskas; AMD-Associated Silent Substitutions in HTRA1 Hijack the Unfolded Protein Response in RPE. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2596.
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© ARVO (1962-2015); The Authors (2016-present)
High Temperature Requirement A1 (HtrA1) is one of the most frequently polymorphic genes in humans with age-related macular degeneration (AMD). Our objective is to understand the role of HtrA1 in the RPE, a site of early and progressive lesions in AMD.
Cultured ARPE-19 cells were stimulated with various doses of tunicamycin in order to inhibit nascent protein maturation. Total lysates were assayed for ER stress markers and HtrA1 expression. To test whether HtrA1 was protective or detrimental to RPE survival during ER stress, RPE cells where HtrA1 was silenced were treated with tunicamycin. To evaluate the effect of AMD-associated variants (rs1049331 and rs2293870, “dSNP”) of HtrA1 on ARPE-19’s unfolded protein response, we re-expressed WT or dSNP HtrA1 into RPE knockdown cells.
When we induced chronic protein misfolding in ARPE-19 cells using tunicamycin, HtrA1 was upregulated just like the canonical chaperones Hsp70 or Hsp90 (n=, p< p<0.001). RPE cells lacking HtrA1 upregulated ER stress markers upon chronic serum deprivation. In response to tunicamycin, HtrA1 knockdown cells failed to resolve ER stress and died (n=6 p<0.001), even in the face of upregulated Hsp70/Hsp90 chaperones. Restoration of WT HtrA1 rescued knockdown cells, but dSNP HtrA1 only partially relieved ER stress-associated death (n=3, p<0.001). Moreover, even though the CMV-driven re-expression of dSNP exceeded the level of HtrA1 in parental/GFP shRNA control cells, dSNP re-expression appeared to lower RPE’s threshold of tolerance for subtoxic doses of tunicamycin (n=3 p<0.001).
HtrA1 is part of RPE’s unfolded protein response, and is essential to survival during ER stress. AMD-associated SNPs in HtrAl, rs1049331 and rs2293870, lower RPE’s tolerance for nutrient deprivation, and enhance sensitivity to ER stress.
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