p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium

Abstract

As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway in cultured human RPE cells, suggesting that p62-mediated autophagy may become the major route to remove impaired proteins under such circumstances. In the present studies, we found that all p62 mRNA variants are abundantly expressed and upregulated by CS induced stress in cultured human RPE cells, yet isoform1 is the major translated form. We also show that p62 silencing exacerbated the CS induced accumulation of damaged proteins, both by suppressing autophagy and by inhibiting the Nrf2 antioxidant response, which in turn, increased protein oxidation. These effects of CS and p62 reduction were further confirmed in mice exposed to CS. We found that over-expression of p62 isoform1, but not its S403A mutant, which lacks affinity for ubiquitinated proteins, reduced misfolded proteins, yet simultaneously promoted an Nrf2-mediated antioxidant response. Thus, p62 provides dual, reciprocal enhancing protection to RPE cells from environmental stress induced protein misfolding and aggregation, by facilitating autophagy and the Nrf2 mediated antioxidant response, which might be a potential therapeutic target against AMD.

Keywords: Aging; Autophagy; Nrf2; Oxidative stress; p62.

Figure 1

p62 mRNA variants are expressed in the RPE and up-regulated by CSE. (A) Schematic representation showing the structure of human p62 mRNA variants, and the positions of the primers used. The C-terminal regions of all three mRNA variants are transcribed from 7 common exons, as shown in the figure. (B) Schematic representation showing the structure of human p62 isoform 1. S403 phosphorylation site, positions of PB1 domain, TRAF6 binding site and UBA domain are indicated in the figure. (C) Ethidium bromide-stained gel showing the PCR products for p62. Total RNA from ARPE-19 cells was reverse transcribed and amplified using primers h-p62T1f and h-p62T1r (lane 1 and 2), or primers h-p62T2f and h-p62T2r (lane 3 and 4). Negative controls, with reverse transcriptase omitted during cDNA preparation, are shown in land 1 and 3. (D) ARPE-19 cells were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. Total RNA was extracted and RT-qPCR was conducted to quantify the mRNA levels of p62 variant 1, and p62 variants 2/3. Expression was normalized to PPIA and graphed as percent of ctrl (DMSO treated). The graph represents the mean values ± SEM (n=3). *p<0.05. (E) ARPE-19 cells were transfected with empty vector (lane 1), p62 v1 (lane 2), or p62 v2/3 (lane 3) plasmids. The whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibody against V5, p62 C-terminus, or beta actin.

Figure 2

Over-expression of p62 variant 2/3 suppresses the endogenous p62 expression in RPE, under stressed conditions. (A) ARPE-19 cells were transfected with empty vector, or p62 v2/3 plasmid, and treated with 125ug/ml CSE or an equal volume of DMSO for 24hrs. The whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibody against p62. (B) Band intensity was quantified using GE Healthcare ImageQuant TL software. Data were normalized for the amount of beta actin in each sample and graphed as percent of ctrl (transfected with ctrl siRNA, DMSO treated). Each point represents the average ± SEM of three independent experiments. Asterisk, p < 0.05.

Figure 3

Effect of p62 silencing on the CS-induced protein damages and autophagic protection in RPE. (A) ARPE-19 cells, after transfection with ctrl or p62 siRNA, were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. Whole cell lysates, prepared in Buffer A, were diluted to 5μg/ml and coated onto the protein carbonyl ELISA plate. Data are presented as the mean±SEM of three independent experiments. *p<0.05. (B) ARPE-19 cells, after transfection with ctrl or p62 siRNA, were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. Whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against p62, polyUb, αB crystallin, or beta actin. (C, D) Quantification of polyubiquitinated proteins and αB crystallin. Band intensities were quantified using GE Healthcare ImageQuant TL software. Data were normalized using beta actin and graphed as percent of ctrl (transfected with ctrl siRNA, DMSO treated). The graphs represent the mean±SEM of three independent experiments. *p<0.05. (E) ARPE-19 cells were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs, in the absence or presence of 50nM BfA, an autophagy inhibitor. Whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against LC3 or beta actin. (F) ARPE-19 cells, after transfection with ctrl or p62 siRNA, were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. Whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibody against LC3. Band intensities were quantified using GE Healthcare ImageQuant TL software. The LC3-II/C3-I ratio was calculated and graphed as percent of ctrl (transfected with ctrl siRNA, DMSO treated). The graph below represents the mean±SEM of three independent experiments. *p<0.05.

Figure 4

p62 silencing reduces autophagosomes. ARPE-19 cells, grown on glass bottom plates, were transfected with ctrl or p62 siRNA and then treated with either 125μg/ml CSE or DMSO for 24hrs. Cells were co-stained with Hoechst 33342 for nuclei (blue) and Cyto-ID (green) for autophagosomes, and viewed with a confocal microscope. Bar=25μm.

Figure 5

Over-expression of p62 rescues RPEs by reducing protein damages, and phosphorylation at S403 is crucial for the protection. ARPE-19 cells were transfected with empty vector or wild-type/mutant p62 plasmids, and treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. (A) Whole cell lysates, prepared in Buffer A, were diluted to 5μg/ml and coated onto the protein carbonyl ELISA plate. Data are presented as mean±SEM of 3 independent experiments. *p<0.05. (B) Whole cell lysates were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against p62, polyUb, αB crystalline or beta actin. (C) Quantification of polyubiquitinated proteins and αB crystallin. Band intensities were quantified using GE Healthcare ImageQuant TL software. Data were normalized using beta actin and graphed as percent of ctrl (transfected with ctrl siRNA, DMSO treated). The graph represents the mean±SEM of three independent experiments. *p<0.05.

Figure 6

Reciprocal regulation of p62 expression and Nrf2 signaling, under basal and stressed conditions. ARPE-19 cells were transfected for p62 silencing (A, B), over-expression of wild type/mutant p62 (C, D), or silencing of Nrf2 or Keap1 (E). After transfection, cells were treated with 125μg/ml CSE or an equal volume of DMSO for 24hrs. Total RNA was extracted for RT-qPCR to quantify the expression of Nqo1, Gclm or p62. Mean values±SEM (n=3 independent experiments) were normalized to PPIA and graphed as percent of ctrl (transfected with ctrl siRNA/DNA, DMSO treated). *p<0.05.

Figure 7

CS-induced protein damage and protective responses in mouse RPE/choroid. 2-mo old WT and Nrf2^−/− mice were raised in a smoking chamber or filtered air environment for 3 weeks and then sacrificed, and eyes were enucleated. (A) Whole cell lysates from RPE/choroid and retina of WT mice were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against p62 or beta actin. (B) Whole cell lysates from RPE/choroid of WT and Nrf2^−/− mice were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against polyUb, LC3, or beta actin. (C) Quantification of polyubiquitinated proteins and LC3. Lane and Band intensity were quantified using GE Healthcare ImageQuant TL software. The amount of polyubiquitinated proteins was normalized using beta actin, and graphed as percent of control (ctrl; WT raised in air). *p<0.05. The LC3-II/LC3-I ratio was calculated, and graphed as percent of control (ctrl; WT raised in air). *p<0.05. (D) Total RNA was extracted from RPE/choroid of WT and Nrf2^−/− mice, and RT-qPCR quantified the expression of p62. Mean values±SEM (n=3 independent experiments) were normalized to PPIA and graphed as percent of control (ctrl; WT raised in air). *p<0.05. (E) Whole cell lysates from RPE/choroid of WT and Nrf2^−/− mice were subjected to 4-12% SDS-PAGE and immunoblotted with antibodies against p62 or beta actin. Band intensity was quantified using GE Healthcare ImageQuant TL software. Data were normalized using beta actin and graphed as percent of control (ctrl; WT raised in air). Data represent the mean±SEM of three independent experiments. *p<0.05. Total RNA was extracted from RPE/choroid of WT and Nrf2^−/− mice, and RT-qPCR quantified the expression of Nqo1 (F) and Gclm (G). Mean values±SEM (n=3 independent experiments) were normalized to PPIA and graphed as percent of control (ctrl; WT raised in air). *p<0.05.