PKC isoform-specific enhancement of capacitative calcium entry in human corneal epithelial cells.
OBJECTIVE
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 Mn2+ 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: delta > betaI > alpha approximately epsilon. Preferential PKCdelta and PKCbeta translocation was in accordance with other results showing that rottlerin and hispidin have the highest efficacy in suppressing EGF-induced CCE augmentation. Furthermore, after PKCbeta and PKCdelta 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 PKCdelta and PKCbeta isoform stimulation. This rank order is based on the findings that either selective knockdown of their expression or exposure to PKCdelta and PKCbeta isoform inhibitors elicited the largest declines in EGF-augmented CCE.
Zhang
Fan
F
Department of Biological Sciences, College of Optometry, State University of New York, New York, New York 10036, USA.
Wen
Quan
Q
Mergler
Stefan
S
Yang
Hua
H
Wang
Zheng
Z
Bildin
Victor N
VN
Reinach
Peter S
PS
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000201:enzymology*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007527:Isoenzymes / Q000502:physiology; D008856:Microscopy, Fluorescence; D009281:Naphthalenes / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D007527:Isoenzymes; D009281:Naphthalenes; D034741:RNA, Small Interfering; C049925:fura-2-am; D015240:Phorbol 12,13-Dibutyrate; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; C058819:calphostin C; D002118:Calcium; D016257:Fura-2