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

Abstract

Background/Aims: The inositol 1,4,5-trisphosphate receptor (IP₃R), a ligand-gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺. Three isoforms of IP₃R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP₃R signalling is involved in the activation of the Ca²⁺-sensitive transcription factors NFAT and CREB. Methods: Each IP₃R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca²⁺ was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. Results: IP₃R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca²⁺ 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 IP₃R-2-knocked down HEK 293A cells. Overexpression of IP₃R-1 could fully compensate for IP₃R-2 knock down to mobilize Ca²⁺ and to activate NFAT. Conclusion: Our results show that the knock down of IP₃R-2 significantly reduced the intracellular Ca²⁺ response of HEK 293 cells. This reduced Ca²⁺ response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca²⁺ signals required for the activation of NFAT are stronger than those required for the activation of CREB.

Transcriptional regulation in the absence of Inositol Trisphosphate Receptor Calcium Signaling

“…Activation of endogenous histamine receptors in HeLa cells showed a 2-fold activation of NFAT in WT cells which is lost in TKO cells (Fig 1B). These observation are in line with published data showing loss of NFAT stimulation by B-cell receptor activation in DT40 TKO cells (2), and reduced NFAT activation in HEK293A cells after siRNA knockdown of type 2 IP3Rs (21). As a control to verify that NFAT was still responsive to Ca 2+ in TKO cells, we tested the effect of the Ca 2+ ionophore ionomycin.…”

“…The different ionophore response in HeLa cells may be due to the lack of RCAN1 changes, but it should be noted that the regulation of CRTC2 is also different in HeLa cells since they lack an upstream kinase (LKB1) needed to maintain phosphorylation of CRTC2 (31). In a previously published study on HEK293A cells it was shown that optimal responses of CREB to Ca 2+ mobilizing hormone requires the addition of vasoactive intestinal peptide (VIP), a cAMP-elevating stimulus (21). Treatment with VIP alone or in combination with ionophore caused a very large activation of CREB in both cell lines which was not significantly different in the TKO cells (Fig 2A & B).…”

Vascular smooth muscle cell phenotype is defined by Ca2+‐dependent transcription factors

“…In contrast, CREB inhibition involves BK Ca -dependent hyperpolarization modulating [Ca 2+ ] i via LTCC channels [35], and this signal will therefore be significantly affected by the initial activation state of VSMCs, their basal [Ca 2+ ] i , and membrane potential, as well as the number of LTCC channels on the membrane. Interestingly, NFAT signalling has recently been shown to directly depend on Ca 2+ release from IP 3 Rs, whereas CREB was generally unaffected by IP 3 R knockdown in HEK cells [94]. This is in contrast to the VSMC data [41], but whether this is attributable to cell differences or whether the previously shown importance of IP 3 Rs for CREB activation is also indirect, similarly to RYRmediated signalling [59], remains unclear.…”

RMP Plays Distinct Roles in the Proliferation of Hepatocellular Carcinoma Cells and Normal Hepatic Cells

“…The mRNA expression of RMP was also examined in human normal cell lines including kidney QBI-293A cells 23, WI-38 cells of a diploid human cell culture line composed of fibroblasts from normal embryonic lung of female 24 and three normal hepatic cell lines of QSG-770125, HL-7702 26 and L02 27. Apparently, all 7 cancer cell lines expressed higher RMP mRNA level than that in 5 normal cell lines (Fig.…”