GATA transcription factors are involved in IgE‐dependent mast cell degranulation by enhancing the expression of phospholipase C‐γ1

Abstract

Mast cell degranulation is a dynamic, highly organized process involving numerous signaling molecules and enzymes. Although the molecular mechanisms underlying antigen-mediated mast cell degranulation have been studied intensively, little is known about the transcriptional control of this process. Here, we show that the hematopoietic transcription factors GATA1 and GATA2 are involved in mast cell degranulation through the control of phospholipase C-c1 (PLC-c1) expression. Knockdown of GATA1 and ⁄ or GATA2 by specific siRNA significantly reduced antigen-induced degranulation and Ca 2+ mobilization in the rat basophilic leukemia cell line RBL-2H3. RT-PCR analyses showed that PLC-c1 expression was significantly decreased by this GATA factor repression. Other GATA factor targets, such as the previously reported a and b subunits of the high-affinity IgE receptor (FceRI), were unaffected. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that GATA factors directly activate PLC-c1 gene transcription through a conserved GATA-binding motif that resides in the 5¢-upstream sequence. Furthermore, we show evidence that the PLC-c1 expression is regulated by GATA2 in mast cells derived from mouse bone marrow. These data indicate that PLC-c1 is a target gene of GATA factors in mast cells and provide evidence that GATA1 and GATA2 control antigen-mediated mast cell degranulation by regulating the expression of PLC-c1.

Critical Roles for PU.1, GATA1, and GATA2 in the Expression of Human FcεRI on Mast Cells: PU.1 and GATA1 Transactivate FCER1A, and GATA2 Transactivates FCER1A and MS4A2

“…These results also demonstrate that PU.1 and GATA1 are involved in FcεRI expression by transactivation of FCER1A and that GATA2 transactivates FCER1A and MS4A2. This observation does not concur with the data from previous studies using siRNA, in which neither GATA1 siRNA nor GATA2 siRNA affected FcεRI expression in BMMCs and/or RBL-2H3 cells (10,11). This discrepancy may be due to specific differences between humans and rodents.…”

“…In our previous studies, PU.1, GATA1, and GATA2 were identified as specific transcription factors that participate in the transcriptional regulation of certain genes expressed in mast cells, including human FCER1A (FcεRIa), mouse Ms4a2 (FcεRIb), mouse c-kit, and human ST2/ IL1RL1 (4,5,7,14,18,19). However, it has also been reported that knockdown of GATA1 and GATA2 by siRNA does not affect FcεRI expression in rodent mast cells and/or a basophilic cell line (10,11).…”

“…Because the degranulation degree parallels the FcεRI expression level, the decreased expression of FcεRI in siRNA-introduced cells can explain this repression of degranulation. Furthermore, it is reported that GATA1 and GATA2 are involved in the expression of several molecules related to the IgE-induced signal transduction of terminally differentiated mast cells (10,11). Therefore, siRNAs for GATA1 and GATA2 may have additional effects targeting the gene expression of intracellular molecules.…”

“…Among three probes, 2683/2654 (probe 1 containing GGATAG, reverse), 2486/2457 (probe 2 containing TGATAGA-TAT), and 2343/2314 (probe 3 containing GGATAA), a specific band shift, which disappeared in the presence of anti-GATA2 Ab, was detected only in the case of probe 2 (Fig. 4B, marked with an asterisk, lanes [10][11][12]. From these data and the results of the reporter assay, we conclude that GATA2 transactivates the MS4A2 promoter with direct binding to the promoter via the TGATA-GATAT sequence at 2476/2467.…”

“…In these previous studies, reporter assays and EMSAs were mainly used to identify and to analyze the role of the transcription factors regulating the promoter activation. In contrast, the involvement of GATA1 and GATA2 in the expression of FcεRI was denied in some studies using mouse bone marrow-derived mast cells (BMMCs) and/or the rat basophilic leukemia cell line, RBL-2H3 (10,11). Therefore, in the current study, we analyzed the involvement of PU.1, GATA1, and GATA2 in FcεRI expression in the human mast cell line LAD2 and primary human mast cells using small interfering RNA (siRNA) and the chromatin immunoprecipitation (ChIP) assay to verify our previous findings.…”