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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

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.
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pone.0134870.g006: 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.

Mentions: These data suggest the importance of PGC-1α for lysosomal activity in RPE cells. If the αvβ5 integrin/FAK/PGC-1α pathway were not sufficiently activated, RPE cells would not efficiently catabolize POS, leading to an abnormal lipid accumulation. Following this, we evaluated the role of PGC-1α in POS degradation. Using Oil Red O staining, we compared intracellular lipid accumulation in cells transfected with PGC-1α siRNA and control siRNA before and after POS treatment. The staining was more intense in ARPE-19 cells whose PGC-1α was silenced than in controls before and after 6 and 12 h of POS treatment (Fig 6A). We also examined the accumulation of peroxidized lipids after POS treatment using BODIPY C11 dye (Fig 6B). The results revealed that PGC-1α silencing approximately doubled the accumulation of peroxidized lipid in POS-treated RPE cells.


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

Roggia MF, Ueta T - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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pone.0134870.g006: 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.
Mentions: These data suggest the importance of PGC-1α for lysosomal activity in RPE cells. If the αvβ5 integrin/FAK/PGC-1α pathway were not sufficiently activated, RPE cells would not efficiently catabolize POS, leading to an abnormal lipid accumulation. Following this, we evaluated the role of PGC-1α in POS degradation. Using Oil Red O staining, we compared intracellular lipid accumulation in cells transfected with PGC-1α siRNA and control siRNA before and after POS treatment. The staining was more intense in ARPE-19 cells whose PGC-1α was silenced than in controls before and after 6 and 12 h of POS treatment (Fig 6A). We also examined the accumulation of peroxidized lipids after POS treatment using BODIPY C11 dye (Fig 6B). The results revealed that PGC-1α silencing approximately doubled the accumulation of peroxidized lipid in POS-treated RPE cells.

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