May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
PKC Alpha and Epsilon Play a Role in EGF-Induced Rat Conjunctival Goblet Cell Proliferation
Author Affiliations & Notes
  • M. A. Shatos
    Dept of Ophthal/Harvard Med Sch, Schepens Eye Research Institute, Boston, Massachusetts
  • R. R. Hodges
    Dept of Ophthal/Harvard Med Sch, Schepens Eye Research Institute, Boston, Massachusetts
  • D. A. Dartt
    Dept of Ophthal/Harvard Med Sch, Schepens Eye Research Institute, Boston, Massachusetts
  • Footnotes
    Commercial Relationships M.A. Shatos, None; R.R. Hodges, None; D.A. Dartt, None.
  • Footnotes
    Support NIH EY09057
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2748. doi:
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      M. A. Shatos, R. R. Hodges, D. A. Dartt; PKC Alpha and Epsilon Play a Role in EGF-Induced Rat Conjunctival Goblet Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2748.

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

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Abstract

Purpose:: To determine whether protein kinase C (PKC) isoforms play a role in EGF-induced proliferation of rat cultured conjunctival goblet cells.

Methods:: For all experiments, cultured P1 rat goblet cells grown to 75% confluence were serum-starved for 24h in RPMI-1640 medium containing 0.5% BSA. PKC inhibitor experiments consisted of treating serum-starved cells with the classic and novel PKC inhibitor, calphostin-C at concentrations ranging from 10-10 to 10-7 M with and without EGF(10-7 M) for 18-20 hr. Using the same treatment protocol, constitutively active PKCα (107 PFU) as well as the dominant negative isoforms of PKCα and PKCε (106 PFU) were transfected into goblet cells by adenoviruses to determine their role in EGF-induced cell proliferation. All treatments were performed at least in triplicate and each experiment was repeated at least four times. The colorimetric WST-8 cell counting assay was used to determine cell proliferation.

Results:: Calphostin C inhibited EGF-stimulated cell proliferation at all concentrations tested. Percent inhibition ranged from 107% at 10-10 M; 111% at 10- 9 M; 95% at 10-8 M and 60% at 10-7 M. Constitutively active PKCα alone significantly increased cell proliferation 52% over basal values. In the same experiments, EGF had no additive effect. The addition of the dominant negative PKC isoforms PKCα and PKCε resulted in a significant decrease of 34% and 60% respectively in EGF-induced goblet cell proliferation.

Conclusions:: EGF-stimulated conjunctival goblet cell proliferation is dependent upon activation of PKC isoforms with both PKCα and PKCε playing important roles.

Keywords: conjunctiva • proliferation • growth factors/growth factor receptors 
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