TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.
Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, we determined, in human corneal epithelial cells, whether or not (i) CCE is dependent upon stimulation of storeoperated channel (SOC) activity, (ii) the canonical transient receptor potential (TRP) protein isoform TRPC4 is a component of such channels, and (iii) suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppressed TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-in-duced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89 and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; 2) EGF-induced CCE fell by 76%; 3) EGF-induced stimulation of SOC activity was eliminated; and 4) EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor.
Yang
Hua
H
College of Optometry, State University of New York, New York 10036, USA.
Mergler
Stefan
S
Sun
Xingcai
X
Wang
Zheng
Z
Lu
Luo
L
Bonanno
Joseph A
JA
Pleyer
Uwe
U
Reinach
Peter S
PS
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000378:metabolism; D004533:Egtazic Acid / Q000737:chemistry; D004594:Electrophysiology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D015151:Immunoblotting; D007473:Ion Channels / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009532:Nickel / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D007473:Ion Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; C022838:nickel chloride; D009532:Nickel; D002118:Calcium; D013936:Thymidine