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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 binding activates αvβ5 integrin/FAK/PGC-1α pathway in ARPE-19 cells.(A) PGC-1α mRNA level in cells was not influenced by the treatment with latex beads (LB). Mean ± SEM, n = 6 per group; two-tailed Student’s t-test; ns, not significant. (B) Upregulation of PGC-1α mRNA by POS treatment was significantly suppressed by pretreatment with RGD peptide. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (C) Time course of PGC-1α mRNA level after POS treatment. (D) Upregulation of PGC-1α mRNA level by POS treatment was significantly suppressed by pretreatment with siRNA against β5 integrin, enhanced by siRNA against CD36 or MerTK, and not influenced by siRNA against Atg5. Mean ± SEM, n = 10 per group, two-tailed Student’s t-test, ****P < 0.0001, **P < 0.01, ***P < 0.001. (E) Upregulation of PGC-1α mRNA level by POS treatment was enhanced by pretreatment with CD36 antibody (2 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, *P < 0.05. (F) Upregulation of PGC-1α mRNA by POS treatment was enhanced by pretreatment with MerTK antibody (3.44 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (G) Upregulation of PGC-1α mRNA by POS treatment was suppressed by pretreatment with FAK inhibitor 14 (500 μM). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001.
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pone.0134870.g002: POS binding activates αvβ5 integrin/FAK/PGC-1α pathway in ARPE-19 cells.(A) PGC-1α mRNA level in cells was not influenced by the treatment with latex beads (LB). Mean ± SEM, n = 6 per group; two-tailed Student’s t-test; ns, not significant. (B) Upregulation of PGC-1α mRNA by POS treatment was significantly suppressed by pretreatment with RGD peptide. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (C) Time course of PGC-1α mRNA level after POS treatment. (D) Upregulation of PGC-1α mRNA level by POS treatment was significantly suppressed by pretreatment with siRNA against β5 integrin, enhanced by siRNA against CD36 or MerTK, and not influenced by siRNA against Atg5. Mean ± SEM, n = 10 per group, two-tailed Student’s t-test, ****P < 0.0001, **P < 0.01, ***P < 0.001. (E) Upregulation of PGC-1α mRNA level by POS treatment was enhanced by pretreatment with CD36 antibody (2 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, *P < 0.05. (F) Upregulation of PGC-1α mRNA by POS treatment was enhanced by pretreatment with MerTK antibody (3.44 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (G) Upregulation of PGC-1α mRNA by POS treatment was suppressed by pretreatment with FAK inhibitor 14 (500 μM). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001.

Mentions: To establish whether the effect was specifically associated with the phagocytosis of POS, we incubated the cells with latex beads used for the assessment of phagocytotic activity [21,22]. In contrast to POS treatment, treatment with latex beads did not increase PGC-1α mRNA (Fig 2A). To eliminate the possibility of other impurities affecting the results, we pretreated ARPE-19 cells for 30 min with RGD peptides, which inhibit POS binding [23] We observed that this pretreatment markedly suppressed the PGC-1α upregulation by POS (Fig 2B). Further, an increase in PGC-1α mRNA levels induced by POS is clearly visible as early as 30 min after POS treatment (Fig 2C). A report has revealed that POS internalization by RPE-J cells occurs after 90 min of the treatment [4]. Another recent study has reported that POS internalization by ARPE-19 and human fetal RPE cells occurs within 30 min after the treatment [15]. The stage of POS phagocytosis (i.e., binding, internalization, or digestion) specifically related to PGC-1α induction by POS remains unclear. To examine the possible scenarios, we first tested the effect of silencing of β5 integrin, CD36, MerTK, and Atg5 by RNA interference (Fig 2D) after confirming their efficiency (S1 Fig). In RPE cells where β5 integrin expression was silenced (i.e., binding was discouraged), PGC-1α upregulation by POS was markedly suppressed. However, when CD36 or MerTK was silenced (i.e., internalization was discouraged), PGC-1α upregulation was markedly enhanced rather than suppressed. PGC-1α upregulation by POS was not affected by Atg5 silencing (i.e., when digestion was discouraged). We also tested the consistency of the results using blocking antibodies against CD36 (2 μg/mL) and MerTK (3.44 μg/mL). In RPE cells pretreated with antibodies against CD36 (Fig 2E) or MerTK (Fig 2F) for 1 h, PGC-1α upregulation was not suppressed but enhanced. These results are consistent with those of a previous report showing that MerTK negatively controls POS binding by limiting the β5 integrin activity [14]. Our results implied that CD36 has similar effects on β5 integrin. If the binding/recognition of POS is associated with the PGC-1α upregulation, FAK, a major intracellular mediator of β5 integrin activation in RPE cells [5,12], may be involved in this signaling pathway. Therefore, we pretreated RPE cells with FAK inhibitor 14 for 30 min and incubated them with and without POS. The increase in PGC-1α mRNA levels caused by POS treatment markedly reduced in RPE cells pretreated with FAK inhibitor compared with controls (Fig 2G), suggesting that FAK is at least partly involved in the upregulation of PGC-1α by POS binding. Finally, we confirmed the effects of siRNAs, blocking antibodies, and other reagents on the binding and internalization of POS (S2 Fig): as expected, both binding and internalization was suppressed by β5 integrin siRNA and RGD peptide while only the internalization was suppressed by siRNAs and antibodies for CD36 and MerTK. Atg5 siRNA did not affect either binding or internalization of POS. FAK inhibitor 14 increased the POS binding significantly but with small extent while it drastically suppressed the internalization.


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

Roggia MF, Ueta T - PLoS ONE (2015)

POS binding activates αvβ5 integrin/FAK/PGC-1α pathway in ARPE-19 cells.(A) PGC-1α mRNA level in cells was not influenced by the treatment with latex beads (LB). Mean ± SEM, n = 6 per group; two-tailed Student’s t-test; ns, not significant. (B) Upregulation of PGC-1α mRNA by POS treatment was significantly suppressed by pretreatment with RGD peptide. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (C) Time course of PGC-1α mRNA level after POS treatment. (D) Upregulation of PGC-1α mRNA level by POS treatment was significantly suppressed by pretreatment with siRNA against β5 integrin, enhanced by siRNA against CD36 or MerTK, and not influenced by siRNA against Atg5. Mean ± SEM, n = 10 per group, two-tailed Student’s t-test, ****P < 0.0001, **P < 0.01, ***P < 0.001. (E) Upregulation of PGC-1α mRNA level by POS treatment was enhanced by pretreatment with CD36 antibody (2 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, *P < 0.05. (F) Upregulation of PGC-1α mRNA by POS treatment was enhanced by pretreatment with MerTK antibody (3.44 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (G) Upregulation of PGC-1α mRNA by POS treatment was suppressed by pretreatment with FAK inhibitor 14 (500 μM). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001.
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pone.0134870.g002: POS binding activates αvβ5 integrin/FAK/PGC-1α pathway in ARPE-19 cells.(A) PGC-1α mRNA level in cells was not influenced by the treatment with latex beads (LB). Mean ± SEM, n = 6 per group; two-tailed Student’s t-test; ns, not significant. (B) Upregulation of PGC-1α mRNA by POS treatment was significantly suppressed by pretreatment with RGD peptide. Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (C) Time course of PGC-1α mRNA level after POS treatment. (D) Upregulation of PGC-1α mRNA level by POS treatment was significantly suppressed by pretreatment with siRNA against β5 integrin, enhanced by siRNA against CD36 or MerTK, and not influenced by siRNA against Atg5. Mean ± SEM, n = 10 per group, two-tailed Student’s t-test, ****P < 0.0001, **P < 0.01, ***P < 0.001. (E) Upregulation of PGC-1α mRNA level by POS treatment was enhanced by pretreatment with CD36 antibody (2 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, *P < 0.05. (F) Upregulation of PGC-1α mRNA by POS treatment was enhanced by pretreatment with MerTK antibody (3.44 μg/mL). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001. (G) Upregulation of PGC-1α mRNA by POS treatment was suppressed by pretreatment with FAK inhibitor 14 (500 μM). Mean ± SEM, n = 6 per group, two-tailed Student’s t-test, ****P < 0.0001.
Mentions: To establish whether the effect was specifically associated with the phagocytosis of POS, we incubated the cells with latex beads used for the assessment of phagocytotic activity [21,22]. In contrast to POS treatment, treatment with latex beads did not increase PGC-1α mRNA (Fig 2A). To eliminate the possibility of other impurities affecting the results, we pretreated ARPE-19 cells for 30 min with RGD peptides, which inhibit POS binding [23] We observed that this pretreatment markedly suppressed the PGC-1α upregulation by POS (Fig 2B). Further, an increase in PGC-1α mRNA levels induced by POS is clearly visible as early as 30 min after POS treatment (Fig 2C). A report has revealed that POS internalization by RPE-J cells occurs after 90 min of the treatment [4]. Another recent study has reported that POS internalization by ARPE-19 and human fetal RPE cells occurs within 30 min after the treatment [15]. The stage of POS phagocytosis (i.e., binding, internalization, or digestion) specifically related to PGC-1α induction by POS remains unclear. To examine the possible scenarios, we first tested the effect of silencing of β5 integrin, CD36, MerTK, and Atg5 by RNA interference (Fig 2D) after confirming their efficiency (S1 Fig). In RPE cells where β5 integrin expression was silenced (i.e., binding was discouraged), PGC-1α upregulation by POS was markedly suppressed. However, when CD36 or MerTK was silenced (i.e., internalization was discouraged), PGC-1α upregulation was markedly enhanced rather than suppressed. PGC-1α upregulation by POS was not affected by Atg5 silencing (i.e., when digestion was discouraged). We also tested the consistency of the results using blocking antibodies against CD36 (2 μg/mL) and MerTK (3.44 μg/mL). In RPE cells pretreated with antibodies against CD36 (Fig 2E) or MerTK (Fig 2F) for 1 h, PGC-1α upregulation was not suppressed but enhanced. These results are consistent with those of a previous report showing that MerTK negatively controls POS binding by limiting the β5 integrin activity [14]. Our results implied that CD36 has similar effects on β5 integrin. If the binding/recognition of POS is associated with the PGC-1α upregulation, FAK, a major intracellular mediator of β5 integrin activation in RPE cells [5,12], may be involved in this signaling pathway. Therefore, we pretreated RPE cells with FAK inhibitor 14 for 30 min and incubated them with and without POS. The increase in PGC-1α mRNA levels caused by POS treatment markedly reduced in RPE cells pretreated with FAK inhibitor compared with controls (Fig 2G), suggesting that FAK is at least partly involved in the upregulation of PGC-1α by POS binding. Finally, we confirmed the effects of siRNAs, blocking antibodies, and other reagents on the binding and internalization of POS (S2 Fig): as expected, both binding and internalization was suppressed by β5 integrin siRNA and RGD peptide while only the internalization was suppressed by siRNAs and antibodies for CD36 and MerTK. Atg5 siRNA did not affect either binding or internalization of POS. FAK inhibitor 14 increased the POS binding significantly but with small extent while it drastically suppressed the internalization.

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