Fig 1.
POS upregulate PGC-1α in RPE cells.
PGC-1α mRNA and protein levels were upregulated in (A) undifferentiated ARPE-19 cells, (B) differentiated ARPE-19 cells, and (C) ex vivo RPE cells, treated with POS for 3 h for evaluation of mRNA and for 6 h for evaluation of protein levels. Morphology of differentiated ARPE-19 cells was confirmed by immunofluorescence for ZO-1 antibody. Mean ± SEM, n = 6–10 per group for mRNA level, n = 3 per group for protein level, two-tailed Student’s t-test, ***P < 0.001, ****P < 0.0001, **P < 0.01, *P < 0.05. Scale bars in the images of undifferentiated and differentiated ARPE-19 cells represent 100 μm and 200 μm, respectively.
Fig 2.
POS binding activates αvβ5 integrin/FAK/PGC-1α pathway in ARPE-19 cells.
(A) PGC-1α mRNA level in cells was not influenced by the treatment with latex beads (LB). Mean ± SEM, n = 6 per group; two-tailed Student’s t-test; ns, not significant. (B) Upregulation of PGC-1α mRNA by POS treatment was significantly suppressed by pretreatment with RGD peptide. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (C) Time course of PGC-1α mRNA level after POS treatment. (D) Upregulation of PGC-1α mRNA level by POS treatment was significantly suppressed by pretreatment with siRNA against β5 integrin, enhanced by siRNA against CD36 or MerTK, and not influenced by siRNA against Atg5. Mean ± SEM, n = 10 per group, two-tailed Student’s t-test, ****P < 0.0001, **P < 0.01, ***P < 0.001. (E) Upregulation of PGC-1α mRNA level by POS treatment was enhanced by pretreatment with CD36 antibody (2 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, *P < 0.05. (F) Upregulation of PGC-1α mRNA by POS treatment was enhanced by pretreatment with MerTK antibody (3.44 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (G) Upregulation of PGC-1α mRNA by POS treatment was suppressed by pretreatment with FAK inhibitor 14 (500 μM). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001.
Fig 3.
POS treatment decreases ROS level and upregulates mitochondrial biogenesis in ARPE-19 cells through PGC-1α.
(A) POS treatment upregulated mRNA levels of anti-oxidant enzymes including GPx1, GPx4, SOD1 and catalase. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ** P < 0.01, *** P < 0.001; ns, not significant. (B) POS treatment downregulated the Intracellular ROS levels evaluated by H2DCFDA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001; ns, not significant. (C) Knockdown Efficacy of PGC-1α siRNA was confirmed by mRNA levels measured by RT-PCR (Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001) and protein levels by western blot analysis (mean ± SEM, n = 3 per group, two-tailed Student’s t-test, **P < 0.01). (D) POS treatment decreased ROS level in cells transfected with control siRNA, but rather increased in those transfected with PGC-1α siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (E) POS treatment upregulated relative mitochondrial DNA content. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (F) Western blot analysis showed the increased protein level of mitochondrial marker prohibitin in cells treated with POS. Mean ± SEM, n = 3 per group, two-tailed Student’s t-test, *P < 0.05. (G) POS treatment upregulated mitochondrial complex I activity in cells transfected with control siRNA, (n = 8 per group, Tukey’s test, P < 0.001), but not in those transfected with PGC-1α siRNA (n = 8 per group, Tukey’s test, P > 0.05).
Fig 4.
POS treatment rescues H2O2-induced SA-β-gal activity through PGC-1α.
ARPE-19 cells were incubated with or without POS for 3 h and then treated with 100 μM H2O2 for 2 h to induce senescence. In cells transfected with control siRNA, POS treatment almost completely rescued SA-β-gal activity induced by H2O2. In contrast, in cells transfected with PGC-1α siRNA, H2O2 induced significantly stronger and more frequent SA-β-gal staining, and POS treatment did not rescue as much as it did in cells transfected with control siRNA. Mean ± SEM, n = 3 per group, ANOVA and Tukey’s test, *P < 0.05, **P < 0.01. Scale bars in large pictures and in insets are 100 μm and 500 μm, respectively.
Fig 5.
PGC-1α regulates lysosomal activity in ARPE-19 cells.
(A) Nuclear immunofluorescence with TFEB antibody was weaker in cells transfected with PGC-1α siRNA compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001; scale bar, 50 μm. (B) In cells transfected with PGC-1α siRNA, mRNA levels of TFEB target genes including galactosidase α (GLA), hexosaminidase A (HEXA), tripeptidyl peptidase I (TPPI), and cathepsin F (CTSF) were downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) In cells pretreated with FAK inhibitor 14, mRNA levels of TFEB target genes including GLA, HEXA, TPPI, and CTSF were downregulated compared to those pretreated with vehicle control. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001. (D) In cells transfected with PGC-1α siRNA, cathepsin D activity was significantly downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ***P < 0.001.
Fig 6.
PGC-1α facilitates lysosomal degradation of POS in ARPE-19 cells.
(A) In cells transfected with PGC-1α siRNA, Oil Red O staining after POS treatment was more intense than those transfected with control siRNA. Scale bar, 200 μm. (B) In cells transfected with PGC-1α siRNA, the accumulation of peroxidized lipids evaluated by BODIPY C11 fluorescence increased after POS treatment compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, ANOVA and Tukey’s test, *P < 0.05.
Fig 7.
Accelerated senescence is observed in RPE of PGC-1α-deficient mice on TEM.
(A, B) The number of melanolysosomes is increased in the RPE of PGC-1α-deficient mice, especially at the basal area. CC; choriocapillaris; scale bar, 5 μm. (C, D) Bruch’s membrane (BM), CC, and basal infoldings of RPE (asterisks). Note that BM of the PGC-1α-deficient mice is thicker with increased electron density than that of the control mice. CC is not well observed in PGC-1α-deficient mice, while it is abundant in the control mice. Loss of truncation of basal infoldings is evident in PGC-1α-deficient mice. Scale bar, 1 μm. (E, F) Magnified view of BM and CC. Scale bar, 500 nm. (G, H) CC is poorly depicted by UEA-I staining in PGC-1α-deficient mice compared to the control mice. CC is well depicted in a dot-like pattern corresponding to the capillaries in WT mice. Scale bar, 5 μm. (I) Magnified views of various type 2 lysosomes in RPE cells of PGC-1α-deficient mice (black arrows). Scale bar, 1 μm.
Fig 8.
Schematic drawing of the pathway shown in the present study.
In RPE cells POS binding activates αvβ5 integrin/FAK/PGC-1α pathway, which confers protections and facilitates lysosomal activity.