The transcription factors NFAT and CREB have different susceptibilities to the reduced Ca2+ responses caused by the knock down of inositol trisphosphate receptor in HEK 293A cells.
OBJECTIVE
The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). Three isoforms of IP(3)R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP(3)R signalling is involved in the activation of the Ca(2+)-sensitive transcription factors NFAT and CREB.


METHODS
Each IP(3)R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca(2+) was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs.


RESULTS
IP(3)R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca(2+) response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP(3)R-2-knocked down HEK 293A cells. Overexpression of IP(3)R-1 could fully compensate for IP(3)R-2 knock down to mobilize Ca(2+) and to activate NFAT.


CONCLUSIONS
Our results show that the knock down of IP(3)R-2 significantly reduced the intracellular Ca(2+) response of HEK 293 cells. This reduced Ca(2+) response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca(2+) signals required for the activation of NFAT are stronger than those required for the activation of CREB.
Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
Guillaume
G
Arguin
Annabelle Z
AZ
Caron
Ghadi
G
Elkoreh
Jean-Bernard
JB
Denault
Gaétan
G
Guillemette
D002118:Calcium / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering
D017362:Cyclic AMP Response Element-Binding Protein; D053496:Inositol 1,4,5-Trisphosphate Receptors; D050778:NFATC Transcription Factors; D034741:RNA, Small Interfering; D002118:Calcium