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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

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.
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pone.0134870.g007: 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.

Mentions: To further elucidate the role of PGC-1α in lipid metabolism and senescence in RPE cells, phenotypes of 6-month-old PGC-1α-deficient mice and age-matched control littermates were compared. We examined the phenotypes of RPE, Bruch’s membrane, and choriocapillaris (the original site of AMD). The retina of PGC-1α-deficient mice demonstrates regular morphology and function [34]. In contrast, our TEM examination revealed an abundant accumulation of melanolysosomes, especially type 2 lysosomes (Fig 7B and 7I vs. 7A) and loss of truncation of basal infoldings (Fig 7D vs. 7C) in RPE cells. Type 2 lysosomes [35] and loss of truncation of basal infoldings [9] are considered associated with aging due to impaired lysosomal activity. We also observed the thickening of the Bruch’s membrane (Fig 7D vs. 7C; Fig 7F vs. 7E) and scarcity of choriocapillaris (Fig 7B vs. 7A) in 6-month-old PGC-1α-deficient mice compared with controls. These phenotypes are also associated with aging RPE cells [1,36–38]. Staining with UEA-I lectin confirmed poor choriocapillaris vasculature in PGC-1α-deficient mice compared with controls (Fig 7H vs. 7G).


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

Roggia MF, Ueta T - PLoS ONE (2015)

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.
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pone.0134870.g007: 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.
Mentions: To further elucidate the role of PGC-1α in lipid metabolism and senescence in RPE cells, phenotypes of 6-month-old PGC-1α-deficient mice and age-matched control littermates were compared. We examined the phenotypes of RPE, Bruch’s membrane, and choriocapillaris (the original site of AMD). The retina of PGC-1α-deficient mice demonstrates regular morphology and function [34]. In contrast, our TEM examination revealed an abundant accumulation of melanolysosomes, especially type 2 lysosomes (Fig 7B and 7I vs. 7A) and loss of truncation of basal infoldings (Fig 7D vs. 7C) in RPE cells. Type 2 lysosomes [35] and loss of truncation of basal infoldings [9] are considered associated with aging due to impaired lysosomal activity. We also observed the thickening of the Bruch’s membrane (Fig 7D vs. 7C; Fig 7F vs. 7E) and scarcity of choriocapillaris (Fig 7B vs. 7A) in 6-month-old PGC-1α-deficient mice compared with controls. These phenotypes are also associated with aging RPE cells [1,36–38]. Staining with UEA-I lectin confirmed poor choriocapillaris vasculature in PGC-1α-deficient mice compared with controls (Fig 7H vs. 7G).

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