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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 regulates lysosomal activity in ARPE-19 cells.(A) Nuclear immunofluorescence with TFEB antibody was weaker in cells transfected with PGC-1α siRNA compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001; scale bar, 50 μm. (B) In cells transfected with PGC-1α siRNA, mRNA levels of TFEB target genes including galactosidase α (GLA), hexosaminidase A (HEXA), tripeptidyl peptidase I (TPPI), and cathepsin F (CTSF) were downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) In cells pretreated with FAK inhibitor 14, mRNA levels of TFEB target genes including GLA, HEXA, TPPI, and CTSF were downregulated compared to those pretreated with vehicle control. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001. (D) In cells transfected with PGC-1α siRNA, cathepsin D activity was significantly downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ***P < 0.001.
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pone.0134870.g005: PGC-1α regulates lysosomal activity in ARPE-19 cells.(A) Nuclear immunofluorescence with TFEB antibody was weaker in cells transfected with PGC-1α siRNA compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001; scale bar, 50 μm. (B) In cells transfected with PGC-1α siRNA, mRNA levels of TFEB target genes including galactosidase α (GLA), hexosaminidase A (HEXA), tripeptidyl peptidase I (TPPI), and cathepsin F (CTSF) were downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) In cells pretreated with FAK inhibitor 14, mRNA levels of TFEB target genes including GLA, HEXA, TPPI, and CTSF were downregulated compared to those pretreated with vehicle control. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001. (D) In cells transfected with PGC-1α siRNA, cathepsin D activity was significantly downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ***P < 0.001.

Mentions: The accumulation of peroxidized lipids and lipofuscin is a hallmark of aged RPE cells [1,6,7,9] and is associated with a decline in lysosomal activity [7,28]. Transcription factor EB (TFEB) is a master regulator of lysosomal activity [29,30]. TFEB and PGC-1α cooperatively regulate lysosomal activity and lipid catabolism in Huntington’s disease mice model [31] and under starvation [32]. We speculated that the αvβ5 integrin/FAK/PGC-1α pathway may be involved in lysosomal activity, facilitating POS metabolism in RPE cells. Furthermore, we observed that when PGC-1α was silenced, nuclear staining of RPE cells with TFEB antibody was markedly weaker than in the cells transfected with control siRNA (Fig 5A). PGC-1α silencing reduced mRNA levels of TFEB target genes encoding galactosidase α, hexosaminidase A, tripeptidyl peptidase I, and cathepsin F (Fig 5B). mRNA levels of these genes were also downregulated by FAK inhibitor 14 (Fig 5C). Moreover, PGC-1α silencing markedly decreased cathepsin D activity (Fig 5D), a major cathepsin involved in the degradation of rhodopsin and POS [6,9,33].


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

Roggia MF, Ueta T - PLoS ONE (2015)

PGC-1α regulates lysosomal activity in ARPE-19 cells.(A) Nuclear immunofluorescence with TFEB antibody was weaker in cells transfected with PGC-1α siRNA compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001; scale bar, 50 μm. (B) In cells transfected with PGC-1α siRNA, mRNA levels of TFEB target genes including galactosidase α (GLA), hexosaminidase A (HEXA), tripeptidyl peptidase I (TPPI), and cathepsin F (CTSF) were downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) In cells pretreated with FAK inhibitor 14, mRNA levels of TFEB target genes including GLA, HEXA, TPPI, and CTSF were downregulated compared to those pretreated with vehicle control. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001. (D) In cells transfected with PGC-1α siRNA, cathepsin D activity was significantly downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ***P < 0.001.
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pone.0134870.g005: PGC-1α regulates lysosomal activity in ARPE-19 cells.(A) Nuclear immunofluorescence with TFEB antibody was weaker in cells transfected with PGC-1α siRNA compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001; scale bar, 50 μm. (B) In cells transfected with PGC-1α siRNA, mRNA levels of TFEB target genes including galactosidase α (GLA), hexosaminidase A (HEXA), tripeptidyl peptidase I (TPPI), and cathepsin F (CTSF) were downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) In cells pretreated with FAK inhibitor 14, mRNA levels of TFEB target genes including GLA, HEXA, TPPI, and CTSF were downregulated compared to those pretreated with vehicle control. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, **P < 0.01, ****P < 0.0001. (D) In cells transfected with PGC-1α siRNA, cathepsin D activity was significantly downregulated compared to those transfected with control siRNA. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ***P < 0.001.
Mentions: The accumulation of peroxidized lipids and lipofuscin is a hallmark of aged RPE cells [1,6,7,9] and is associated with a decline in lysosomal activity [7,28]. Transcription factor EB (TFEB) is a master regulator of lysosomal activity [29,30]. TFEB and PGC-1α cooperatively regulate lysosomal activity and lipid catabolism in Huntington’s disease mice model [31] and under starvation [32]. We speculated that the αvβ5 integrin/FAK/PGC-1α pathway may be involved in lysosomal activity, facilitating POS metabolism in RPE cells. Furthermore, we observed that when PGC-1α was silenced, nuclear staining of RPE cells with TFEB antibody was markedly weaker than in the cells transfected with control siRNA (Fig 5A). PGC-1α silencing reduced mRNA levels of TFEB target genes encoding galactosidase α, hexosaminidase A, tripeptidyl peptidase I, and cathepsin F (Fig 5B). mRNA levels of these genes were also downregulated by FAK inhibitor 14 (Fig 5C). Moreover, PGC-1α silencing markedly decreased cathepsin D activity (Fig 5D), a major cathepsin involved in the degradation of rhodopsin and POS [6,9,33].

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