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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 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.
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pone.0134870.g001: 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.

Mentions: We first tested whether the effect of POS treatment on PGC-1α expression in cultured RPE cells was equivalent under different conditions. The treatment increased PGC-1α mRNA and protein levels in undifferentiated ARPE-19 cells (Fig 1A). We observed the same effect in differentiated ARPE-19 cells (Fig 1B) as well as in ex vivo RPE culture (Fig 1C). Here, we used undifferentiated ARPE-19 cells to investigate PGC-1α-related functions in RPE cells.


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

Roggia MF, Ueta T - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134870.g001: 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.
Mentions: We first tested whether the effect of POS treatment on PGC-1α expression in cultured RPE cells was equivalent under different conditions. The treatment increased PGC-1α mRNA and protein levels in undifferentiated ARPE-19 cells (Fig 1A). We observed the same effect in differentiated ARPE-19 cells (Fig 1B) as well as in ex vivo RPE culture (Fig 1C). Here, we used undifferentiated ARPE-19 cells to investigate PGC-1α-related functions in 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