May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Rac1 Activation by vß5 Integrin Receptor Signaling Is Required for OS Phagocytosis by the Retinal Pigment Epithelium
Author Affiliations & Notes
  • E.F. Nandrot
    Dyson Institute/Ophthalmology, Weill Medical College of Cornell University, New York, NY, United States
  • S.C. Finnemann
    Dyson Institute/Ophthalmology, Weill Medical College of Cornell University, New York, NY, United States
  • Footnotes
    Commercial Relationships  E.F. Nandrot, None; S.C. Finnemann, None.
  • Footnotes
    Support  NIH grant EY14184, RPB, Kirchgessner Foundation
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 942. doi:
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      E.F. Nandrot, S.C. Finnemann; Rac1 Activation by vß5 Integrin Receptor Signaling Is Required for OS Phagocytosis by the Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):942.

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

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Abstract

Abstract: : Purpose: To determine the function of actin-regulating Rho family GTPases in OS phagocytosis by RPE and their dependence on RPE phagocytic receptors. Methods: Laser scanning confocal fluorescence microscopy and immunoblotting of RhoA, Rac1, and Cdc42 were used to determine GTPase expression and localization in RPE. RPE cell lines and primary RPE prepared from 12 to 16 day old mutant mice and rats of different strains that each lack one of the RPE phagocytic receptors and of background matched wild-type animals were grown in culture and challenged with isolated outer segment fragments (OS) in the presence or absence of GTPase inhibitors. OS binding and internalization were quantified using fluorescence scanning. Purified recombinant fusion proteins that bind to GTP-loaded (active) but not to GDP-loaded (inactive) GTPases were used to quantitatively isolate active GTPases from RPE lysates. Results: Rho family GTPases, which regulate actin-dependent processes, were abundant in RPE. OS challenge of RPE in culture increased the GTP-bound fraction of Rac1 but not of RhoA or Cdc42 in RPE cells suggesting specific activation of Rac1 during OS phagocytosis. Moreover, Rac1 colocalized with internalized OS in RPE. Bacterial Toxin B (which blocks all three Rho GTPases) but not C3 transferase (which selectively blocks RhoA) inhibited internalization of bound OS. Furthermore, transient expression in RPE of GDP-locked Rac1 (which specifically blocks Rac1) abolished OS internalization. Strikingly, specific surface ligation of αvß5 integrin activated Rac1 with similar kinetics and efficiency as OS challenge. Furthermore, Rac1 activation in response to OS challenge was absent in ß5 knockout RPE lacking αvß5 integrin. In contrast, Rac1 activation by OS was preserved in mutant RPE lacking receptors that mediate OS engulfment, CD36 and MerTK. Conclusions: Rac1 activation is a prerequisite for the engulfment of bound OS by RPE. Furthermore, we show that αvß5 integrin receptor ligation at the apical, phagocytic RPE surface is both necessary and sufficient to activate Rac1. Since OS phagocytic challenge efficiently activates Rac1 independently of CD36 and MerTK, we suggest that Rac1 functions upstream or independent of these internalization receptors in RPE phagocytosis.

Keywords: signal transduction • retinal pigment epithelium • phagocytosis and killing 
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