Limits...
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 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526642&req=5

pone.0134870.g004: 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.

Mentions: We also evaluated the effects of POS and PGC-1α on SA-β-gal activity (Fig 4). We induced ARPE-19 cell senescence by incubation with 100 μM H2O2 for 2 h and conducted SA-β-gal staining 24 h later [19]. The 3-h POS pretreatment almost completely rescued H2O2-induced senescence in RPE cells transfected with control siRNA. In contrast, PGC-1α silencing aggravated H2O2-induced senescence and markedly suppressed the counteracting effect of POS (Fig 4).


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

Roggia MF, Ueta T - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134870.g004: 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.
Mentions: We also evaluated the effects of POS and PGC-1α on SA-β-gal activity (Fig 4). We induced ARPE-19 cell senescence by incubation with 100 μM H2O2 for 2 h and conducted SA-β-gal staining 24 h later [19]. The 3-h POS pretreatment almost completely rescued H2O2-induced senescence in RPE cells transfected with control siRNA. In contrast, PGC-1α silencing aggravated H2O2-induced senescence and markedly suppressed the counteracting effect of POS (Fig 4).

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