May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
An Important Regulatory Role for PKC in Lens Cell Differentiation
Author Affiliations & Notes
  • S. Das
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • M. E. Barnett
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • J. R. McCully
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • G. A. Zampighi
    Neurobiology, University of California Los Angeles, Los Angeles, California
  • L. H. Willard
    Kansas State University, Manhattan, Kansas
    Diagnostic Medicine Pathobiology,
  • D. J. Takemoto
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • Footnotes
    Commercial Relationships S. Das, None; M.E. Barnett, None; J.R. McCully, None; G.A. Zampighi, None; L.H. Willard, None; D.J. Takemoto, None.
  • Footnotes
    Support EYRO1-13421 to DJT and EYRO1-040110 to GZ
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4214. doi:
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      S. Das, M. E. Barnett, J. R. McCully, G. A. Zampighi, L. H. Willard, D. J. Takemoto; An Important Regulatory Role for PKC in Lens Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4214.

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

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Abstract

Purpose:: The purpose of this study was to determine if protein kinase Cγ (PKCγ) is involved in the regulated loss of Cx43 during lens epithelial cell differentiation to fiber cells.

Methods:: N/N 1003A rabbit lens epithelial cells were treated with the PKC activator, TPA at 400nM, for up to 48 hours and Western blotting was performed to quantitate PKCγ, Cx43 and Cx50 protein levels. The distribution of Cx43 in the control (b6129pf21j100903) and PKCγ knockout (B6;129p-Prkcctm1St1) mouse lenses was determined from dissected epithelial, cortical, and nuclear regions. Confocal microscopy was used to determine the location of PKCγ, Cx43 and Cx50. Light and electron microscopy were utilized to examine lens structure.

Results:: Long-term exposure of lens epithelial cells in culture to TPA resulted in the degradation of Cx43 but not Cx50. Since hyper-phosphorylation of Cx43 has been shown to result in the down-regulation of Cx43 we examined the effects of a PKCγ knockout mouse, where phosphorylation of Cx43 by PKCγ does not occur. In normal mouse lenses, PKCγ was found in the epithelial, cortical, and nuclear regions of the whole lens but was absent from the knockout mice. In the wild type mice Cx43 was found only in the epithelial layers. In contrast, in the PKCγ knockout mouse lens, Cx43 persisted into the inner, cortical, and nuclear regions of the lens. The location of Cx50 and Cx46 were identical in both wild type and knockout mice. Light and electron microscopy demonstrated that in the bow region the characteristic V-shape arrangement of nuclei was disrupted in the knockout mice. However, fiber cell differentiation appeared otherwise normal despite the abnormal and persistent distribution of Cx43 in the knockout mouse lens.

Conclusions:: PKCγ is required for the degradation of Cx43 during lens differentiation. The loss of PKCγ does not alter Cx50 or Cx46 levels. The presence of the hyperphosphorylation sites at residues 363 - 375 of Cx43, not found in Cx50, are required for the PKCγ-catalyzed loss of Cx43. This would not occur in the absence of PKCγ as observed in the knockout mice.

Keywords: differentiation • phosphorylation • gene/expression 
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