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Influence of Hsp90 and HDAC inhibition and tubulin acetylation on perinuclear protein aggregation in human retinal pigment epithelial cells.

Ryhänen T, Viiri J, Hyttinen JM, Uusitalo H, Salminen A, Kaarniranta K - J. Biomed. Biotechnol. (2010)

Bottom Line: We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells.However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation.These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Eastern Finland, 70211 Kuopio, Finland.

ABSTRACT
Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

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Related in: MedlinePlus

(a) Total proteins (20 μg) of whole cell extracts were examined by western blot using antibodies against LC3 of control (med) cells, and cells exposed 0.25 μM geldanamycin (GA) or 5 μM MG-132 (MG) or their combination  for 6, 12, and 24 hours. Tubulin was used to check the equal loading of proteins.  (b) Quantifications of western blots and LC3 II/I ratio.
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fig4: (a) Total proteins (20 μg) of whole cell extracts were examined by western blot using antibodies against LC3 of control (med) cells, and cells exposed 0.25 μM geldanamycin (GA) or 5 μM MG-132 (MG) or their combination for 6, 12, and 24 hours. Tubulin was used to check the equal loading of proteins. (b) Quantifications of western blots and LC3 II/I ratio.

Mentions: A robust elevation of Hsp70 protein expression was seen in response to MG-132 or geldanamycin exposures (Figure 3). Hsp90 levels were not markedly affected by the treatments. Abrogation of proteasome-mediated protein degradation caused a clear increase in the amount of Hsp70 which has a cytoprotective capacity in the MG-132 -treated ARPE-19 cells [29]. The classical transcriptional heat-shock gene induction is attributable to the activation of HSF1 transcription factor [32]. In the activation process Hsp90 and Hsp70 dissociate from HSF1 transcription factor [33, 34]. Geldanamycin has been shown to bind Hsp90, to inhibit its function and to elicit the Hsp90 client protein degradation in proteasomes [35, 36]. In line with previous studies [37–39], the geldanamycin was found to trigger a strong expression of Hsp70, while the Hsp90 response remained weaker. The response is likely mediated through HSF1 transcriptional activation [33, 34]. The increase in the amount of inducible Hsp70 might be one of the regulators suppressing the proteasome inhibitor-induced aggregation process [29], when Hsp90 is simultaneously inhibited. It has also been documented that a Hsp90 inhibitor may prevent the aggregation of protein by regulating client protein posttranslational modifications [40]. Since Hsp90 inhibition has been reported to trigger autophagy clearance [41, 42], we wished to analyze the autophagy induction marker LC3 I/II levels in ARPE cells treated with geldanamycin or MG-132 solely or both together for 6, 12, and 24 hours. Our findings clearly show that proteasome inhibition mildly induced autophagy, when related to LC3 II levels, but this was not involved in geldanamycin treatments (Figure 4). This indicates that autophagy clearance is not implicated in the suppression of protein aggregation during Hsp90 and proteasome inhibition in the ARPE-19 cells.


Influence of Hsp90 and HDAC inhibition and tubulin acetylation on perinuclear protein aggregation in human retinal pigment epithelial cells.

Ryhänen T, Viiri J, Hyttinen JM, Uusitalo H, Salminen A, Kaarniranta K - J. Biomed. Biotechnol. (2010)

(a) Total proteins (20 μg) of whole cell extracts were examined by western blot using antibodies against LC3 of control (med) cells, and cells exposed 0.25 μM geldanamycin (GA) or 5 μM MG-132 (MG) or their combination  for 6, 12, and 24 hours. Tubulin was used to check the equal loading of proteins.  (b) Quantifications of western blots and LC3 II/I ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2963810&req=5

fig4: (a) Total proteins (20 μg) of whole cell extracts were examined by western blot using antibodies against LC3 of control (med) cells, and cells exposed 0.25 μM geldanamycin (GA) or 5 μM MG-132 (MG) or their combination for 6, 12, and 24 hours. Tubulin was used to check the equal loading of proteins. (b) Quantifications of western blots and LC3 II/I ratio.
Mentions: A robust elevation of Hsp70 protein expression was seen in response to MG-132 or geldanamycin exposures (Figure 3). Hsp90 levels were not markedly affected by the treatments. Abrogation of proteasome-mediated protein degradation caused a clear increase in the amount of Hsp70 which has a cytoprotective capacity in the MG-132 -treated ARPE-19 cells [29]. The classical transcriptional heat-shock gene induction is attributable to the activation of HSF1 transcription factor [32]. In the activation process Hsp90 and Hsp70 dissociate from HSF1 transcription factor [33, 34]. Geldanamycin has been shown to bind Hsp90, to inhibit its function and to elicit the Hsp90 client protein degradation in proteasomes [35, 36]. In line with previous studies [37–39], the geldanamycin was found to trigger a strong expression of Hsp70, while the Hsp90 response remained weaker. The response is likely mediated through HSF1 transcriptional activation [33, 34]. The increase in the amount of inducible Hsp70 might be one of the regulators suppressing the proteasome inhibitor-induced aggregation process [29], when Hsp90 is simultaneously inhibited. It has also been documented that a Hsp90 inhibitor may prevent the aggregation of protein by regulating client protein posttranslational modifications [40]. Since Hsp90 inhibition has been reported to trigger autophagy clearance [41, 42], we wished to analyze the autophagy induction marker LC3 I/II levels in ARPE cells treated with geldanamycin or MG-132 solely or both together for 6, 12, and 24 hours. Our findings clearly show that proteasome inhibition mildly induced autophagy, when related to LC3 II levels, but this was not involved in geldanamycin treatments (Figure 4). This indicates that autophagy clearance is not implicated in the suppression of protein aggregation during Hsp90 and proteasome inhibition in the ARPE-19 cells.

Bottom Line: We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells.However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation.These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Eastern Finland, 70211 Kuopio, Finland.

ABSTRACT
Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

Show MeSH
Related in: MedlinePlus