June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Hypoxia alters the polarized movement, expression, and secretion of proteins in retinal pigmented epithelial (RPE) cells by inhibiting retromer function
Author Affiliations & Notes
  • Philip Mzyk
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • Steven Nagar
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • Mary Christine McGahan
    Molecular Biomedical Sciences, North Carolina State University, Raleigh, NC
  • Footnotes
    Commercial Relationships Philip Mzyk, None; Steven Nagar, None; Mary McGahan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2331. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Philip Mzyk, Steven Nagar, Mary Christine McGahan; Hypoxia alters the polarized movement, expression, and secretion of proteins in retinal pigmented epithelial (RPE) cells by inhibiting retromer function. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2331.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: Retinal pigmented epithelial (RPE) cells are unique in that their apical surface is in direct contact with the photoreceptor outer segments and the interphotoreceptor matrix. This unique architecture requires tight regulation of polarized protein movement within RPE cells. Retromer regulates the polarized movement of proteins from the trans Golgi network to apical or basolateral surfaces for secretion. Hypoxia occurs when the choroidal blood supply to the RPE is disrupted. We have evidence that hypoxia alters the polarized movement, expression, and secretion of proteins in retinal pigmented epithelial (RPE) cells by inhibiting retromer function.

Methods: RPE were isolated from canine eyes and grown to confluence. Cells were then dispersed on either 6 well tissue culture plates, or onto 6-well Millicell® plates with the apical and basolateral sides bathed separately. Cells were placed under hypoxic (0.5% O2) conditions for 24 and 48 hours, with corresponding cells placed under normoxic conditions (21% O2) as a control. Cells were also transfected with siRNA to knockdown retromer function and then exposed to either hypoxic or normoxic conditions. Lysates and cell-conditioned media (CCM) were collected and analyzed by immunoblotting for retromer and known polarized proteins.

Results: We found that oxygen levels control polarized secretion of proteins in RPE cells. Specifically, in polarized, tight junctional RPE cells 4.5 times more APP was secreted basolaterally versus apically. Under hypoxic conditions APP secretion was decreased in both the apical (94%) and basolateral (72%) direction; while still maintaining a 4 fold higher level of APP secretion in the basolateral vs. apical direction. Additionally, hypoxia decreased retromer levels in polarized RPE cell lysates by 89%. Significantly, when retromer was knocked down with siRNA in non-polarized RPE cells, APP secretion is reduced an average of 38% in both normoxic and hypoxic conditions.

Conclusions: Hypoxia alters polarized protein localization and secretion and this is due in part to retromer inhibition. Understanding how hypoxia affects protein processing, polarized localization, and secretion is vital to our comprehension of RPE pathophysiology.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×