PKC isoform-specific enhancement of capacitative calcium entry in human corneal epithelial cells

Fan Zhang, Quan Wen, Stefan Mergler, Hua Yang, Zheng Wang, Victor N. Bildin, Peter S. Reinach

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

PURPOSE. To determine in human corneal epithelial cells (HCECs) the role of protein kinase C (PKC) in mediating epidermal growth factor (EGF)-induced stimulation of store-operated channel (SOC) activity and capacitative calcium entry (CCE). METHODS. Single-cell Ca2+ fluorescence imaging of fura2-loaded HCECs was used to evaluate CCE. PKC translocation induced by EGF or PDBu was monitored by Western blot analyses of four different subcellular fractions. Plasma membrane Ca2+ influx was measured by Mn 2+ quench rates of fura2-fluorescence. The whole-cell patch clamp configuration was used to determine the SOC activation induced by EGF. RESULTS. EGF-induced increases in SOC currents through PKC stimulation, since calphostin C inhibited this response. To determine which PKC isoforms mediated EGF-induced increases in CCE, the PKC isoform enrichment of a plasma membrane-containing fraction was determined. From 5 to 30 minutes, its rank order of enrichment was: δ > β1 > α∼ε. Preferential PKCδ and PKCβ translocation was in accordance with other results showing that rottlerin and hispidin have the highest efficacy in suppressing EGF-induced CCE augmentation. Furthermore, after PKCβ and PKCδ siRNA knockdown of gene and protein expression, declines in EGF-induced increases in CCE matched those obtained after exposure to a corresponding selective PKC isoform inhibitor. CONCLUSIONS. EGF-induced PKC stimulation in HCECs mediates SOC activation. This response contributes to CCE, which preferentially depends on PKCδ and PKCβ isoform stimulation. This rank order is based on the findings that either selective knockdown of their expression or exposure to PKCδ and PKCβ isoform inhibitors elicited the largest declines in EGF-augmented CCE.

Original languageEnglish
Pages (from-to)3989-4000
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number9
DOIs
StatePublished - Sep 2006
Externally publishedYes

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