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C. Mayo, C. Rich, V. Trinkaus-Randall; Corneal Epithelial Cells Express a Truncated P2X7 Receptor. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4613.
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The P2X7 receptor is an ATP activated cation channel. It has pivotal roles in inflammatory and apoptotic processes as well as the ability to form large, non-specific pores. We have shown that P2X7 is critical for proper stromal ultrastructure. Our goal was to determine the role of P2X7 in corneal epithelium, a tissue that does not normally undergo apoptosis.
In calcium studies, transformed human corneal limbal epithelial cells (HCLE) were stimulated with BzATP in the presence or absence of extracellular Ca2+, Mg2+, or Zn2+ using a flow through apparatus and imaged on a Zeiss LSM510 microscope. Apoptosis was assessed after BzATP treatment using a fluorescent caspase-3 activation kit and pore formation was monitored by dye uptake. Expression of P2X7 was determined by Western and Northern blot analyses and PCR using variant or exon specific primers.
Treatment of HCLEs with BzATP led to an increase in intracellular Ca2+ that was inhibited by the removal of extracellular Ca2+ and enhanced by the removal of extracellular Mg2+ or Zn2+. This behavior is indicative of the P2X7 receptor. Prolonged stimulation with BzATP did not induce apoptosis or lead to large pore formation, in contrast to IMR90 cells that were used as a control. Membrane blebbing upon BzATP treatment was not observed in cells imaged in an environmental chamber. Additionally, treatment with BzATP led to phosphorylation of ERK. Western blot analysis showed that HCLEs express a truncated protein while PCR of genomic DNA indicated that all exons are present. PCR with variant specific primers confirmed that a variant is expressed and Northern blot analysis revealed a truncated mRNA transcript.
Corneal epithelial cells express a truncated P2X7 receptor that lacks the ability to form the large pores usually associated with prolonged activation. Stimulation with BzATP causes canonical Ca2+ responses, but does not induce apoptosis. Western and Northern blot analyses confirmed the presence of spliced mRNA and truncated P2X7 protein in HCLEs. A truncated C-terminus and loss-of-function polymorphisms in the C-terminus have also been reported in cancer cells, supporting the pro-survival role observed in corneal epithelium. These results provide new insights into the expression of P2X7 in corneal epithelium.
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