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αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

Roggia MF, Ueta T - PLoS ONE (2015)

Bottom Line: We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity.The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by photoreceptor outer segments (POS) and its effects on retinal pigment epithelium (RPE).

Methods: PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK), and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK) were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.

Results: POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.

Conclusions: The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0134870.g003: 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).

Mentions: PGC-1α has been known as a master regulator of mitochondrial biogenesis [24,25] and a potent suppressor of oxidative stress [26]. Increased PGC-1α expression in the muscle rescues age-related muscle wasting and metabolic decline in mice [27]. Therefore, we hypothesized that POS-induced PGC-1α upregulation increased mitochondrial biogenesis and decreased ROS production and conferred overall protective effects. We observed an increase in mRNA for the antioxidant enzymes glutathione peroxidase 1 (GPx1), GPx4, superoxide dismutase 1, and catalase (Fig 3A). The overall ROS level decreased in POS-treated RPE cells (Fig 3B). To investigate if the decrease in ROS depends on PGC-1α upregulation, we used PGC-1α siRNA after confirming its efficiency in ARPE-19 cells (Fig 3C). When PGC-1α was silenced by siRNA, POS treatment increased the ROS level in RPE cells (Fig 3D). This result indicates that PGC-1α may have an important role in POS-induced ROS reduction. POS treatment also increased the relative levels of mitochondrial DNA compared with nuclear DNA (Fig 3E). The level of prohibitin, a mitochondrial marker protein, also increased after POS treatment (Fig 3F), suggesting an increased mitochondrial volume. Functionally, POS treatment upregulated mitochondrial complex I activity, which was also abrogated by PGC-1α silencing (Fig 3G).


αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

Roggia MF, Ueta T - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526642&req=5

pone.0134870.g003: 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).
Mentions: PGC-1α has been known as a master regulator of mitochondrial biogenesis [24,25] and a potent suppressor of oxidative stress [26]. Increased PGC-1α expression in the muscle rescues age-related muscle wasting and metabolic decline in mice [27]. Therefore, we hypothesized that POS-induced PGC-1α upregulation increased mitochondrial biogenesis and decreased ROS production and conferred overall protective effects. We observed an increase in mRNA for the antioxidant enzymes glutathione peroxidase 1 (GPx1), GPx4, superoxide dismutase 1, and catalase (Fig 3A). The overall ROS level decreased in POS-treated RPE cells (Fig 3B). To investigate if the decrease in ROS depends on PGC-1α upregulation, we used PGC-1α siRNA after confirming its efficiency in ARPE-19 cells (Fig 3C). When PGC-1α was silenced by siRNA, POS treatment increased the ROS level in RPE cells (Fig 3D). This result indicates that PGC-1α may have an important role in POS-induced ROS reduction. POS treatment also increased the relative levels of mitochondrial DNA compared with nuclear DNA (Fig 3E). The level of prohibitin, a mitochondrial marker protein, also increased after POS treatment (Fig 3F), suggesting an increased mitochondrial volume. Functionally, POS treatment upregulated mitochondrial complex I activity, which was also abrogated by PGC-1α silencing (Fig 3G).

Bottom Line: We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity.The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by photoreceptor outer segments (POS) and its effects on retinal pigment epithelium (RPE).

Methods: PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK), and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK) were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.

Results: POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.

Conclusions: The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

No MeSH data available.


Related in: MedlinePlus