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TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2005 Sep 16; Vol. 280 (37), pp. 32230-7. Date of Electronic Publication: 2005 Jul 20. - Publication Year :
- 2005
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Abstract
- 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.
- Subjects :
- Blotting, Western
Calcium metabolism
Cell Proliferation
Cells, Cultured
Cornea metabolism
Egtazic Acid chemistry
Electrophysiology
Humans
Immunoblotting
Microscopy, Fluorescence
Nickel pharmacology
Patch-Clamp Techniques
RNA, Messenger metabolism
RNA, Small Interfering metabolism
Reverse Transcriptase Polymerase Chain Reaction
TRPC Cation Channels
Thymidine metabolism
Time Factors
Transfection
Cornea cytology
Epidermal Growth Factor metabolism
Epithelial Cells cytology
Ion Channels genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 280
- Issue :
- 37
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 16033767
- Full Text :
- https://doi.org/10.1074/jbc.M504553200