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Dhirendra P Singh, Nigar Fatma, Bhavana Chhunchha, W Daniel Stamer, Eri Kubo; Replicative Senescence of Trabecular Meshwork Cells Is Associated With Loss Of Prdx6 By Sumoylation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3678.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress-induced aberrant Sumoylation of proteins has been found in initiation of replicative senescence. Prdx6 is Sumoylated by Sumo (Small Ubiquitin-like Modifier)-1, resulting in loss of Prdx6 activity. Using primary cultures of human TM cells as a model, we investigated Sumoylation status in glaucomatous or aging cells, and the effect of oxidative stress-induced aberrant Sumoylation signaling on Prdx6 regulation.
Cultured TM cells from normal and glaucomatous human subjects of different ages, and TM cells (Fresh isolates) in complete Opti-MEM medium with H2O2 (0 to 200μM) for variable times were used. We assessed the status of Sumoylation and its effect on Prdx6 expression, as well as ECM proteins, senescence markers p16 and p21 protein, and mRNA by Western analysis, RT-PCR and Epiquick Sumoylation Assay Kit. Cells transfected with either pGFP-Prdx6 or SiRNA, pGFP-Sumo1, pSenp1 or vector constructs. Stably/transiently transfected TM cells exposed to H2O2 were used to assess effect on cell viability and ROS levels by MTS and H2-DCFH-DA dye assays. Transfectants were stained for SA-βgal activity to assess the level of senescence.
Glaucomatous TM cells, late passage (>P8) TM cells and cells from older subjects [(45 or 64 years (y)] had increased Sumoylation of most proteins compared to TM cells from younger subjects (4 and 24y ), consistent with increased ROS, Sumo1 and decreased Senp1 expression. Higher oxidative load enhanced Sumoylation of most proteins, leading to loss of cell integrity. Cells overexpressing Sumo1 showed reduced Prdx6 expression. They had increased expression of TGFβ1/2 and ECM proteins; αsm-actin, fibronectin, thrombospondin1, TGase2, PAI1, tropomyocin, and p16, and p21. Glaucomatous and TM cells of late passage and cells overexpressing Sumo1 displayed increased ROS and in SA-βgal activity with decreased proliferation/viability against oxidative stress. An extrinsic supply of Senp1 or Prdx6 abated senescence, indicating aberrant expression of Sumo1 accelerated senescence.
Aberrant Sumoylation signaling is involved in dysregulating protein function in glaucomatous or TM cells facing oxidative stress, leading to senescence and pathobiology. Since loss of Prdx6 is causally linked to aberrant Sumoylation and etiopathogenesis, development of Prdx6 expression-based therapeutics may delay or prevent TM cell dysfunction.
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