Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells

  1. Tuomas Ryhänen1,†,
  2. Juha M. T. Hyttinen1,†,
  3. Jürgen Kopitz2,
  4. Kirsi Rilla3,
  5. Erkki Kuusisto4,
  6. Eliisa Mannermaa5,
  7. Johanna Viiri1,
  8. Carina I. Holmberg6,
  9. Ilkka Immonen7,
  10. Seppo Meri8,
  11. Jussi Parkkinen9,
  12. Eeva-Liisa Eskelinen10,
  13. Hannu Uusitalo1,
  14. Antero Salminen4,
  15. Kai Kaarniranta1,*

Article first published online: 6 NOV 2008

DOI: 10.1111/j.1582-4934.2008.00577.x

Issue

Journal of Cellular and Molecular Medicine

Journal of Cellular and Molecular Medicine

Volume 13, Issue 9b, pages 3616–3631, September 2009

Additional Information

How to Cite

Ryhänen, T., Hyttinen, J. M. T., Kopitz, J., Rilla, K., Kuusisto, E., Mannermaa, E., Viiri, J., Holmberg, C. I., Immonen, I., Meri, S., Parkkinen, J., Eskelinen, E.-L., Uusitalo, H., Salminen, A. and Kaarniranta, K. (2009), Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells. Journal of Cellular and Molecular Medicine, 13: 3616–3631. doi: 10.1111/j.1582-4934.2008.00577.x

Author Information

  1. 1

    Department of Ophthalmology, University of Kuopio, Kuopio, Finland

  2. 2

    Institute of Molecular Pathology, Medical Faculty of the University of Heidelberg, Heidelberg, Germany

  3. 3

    Department of Anatomy, University of Kuopio, Kuopio, Finland

  4. 4

    Department of Neuroscience and Neurology, University of Kuopio, Kuopio, Finland

  5. 5

    Department of Pharmaceutics, University of Kuopio, Kuopio, Finland

  6. 6

    Molecular and Cancer Biology Program, Institute of Biomedicum, University of Helsinki, Helsinki, Finland

  7. 7

    Department of Ophthalmology, Helsinki University Hospital, Helsinki, Finland

  8. 8

    Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland

  9. 9

    Department of Computer Science and Statistics, University of Joensuu, Joensuu, Finland

  10. 10

    Department of Biological and Environmental Sciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland

  1. †

    These authors contributed equally to this work.

* Correspondence to: K. KAARNIRANTA, Department of Ophthalmology, University of Kuopio, P.O. BOX 1627, 70211 Kuopio, Finland. Tel.: +358-17-162015 Fax: +358-17-162048 E-mail: kaarnira@messi.uku.fi

Publication History

  1. Issue published online: 29 JAN 2010
  2. Article first published online: 6 NOV 2008
  3. Received: May 20, 2008; Accepted: October 20, 2008

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Keywords:

  • autophagosome;
  • Hsp70;
  • lysosome;
  • proteasome;
  • proteolysis;
  • retinal pigment epithelium

Abstract

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

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