The transcription factor Zeb2 regulates signaling in mast cells

Abstract

Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a zinc finger transcription factor, regulates both early and late mast cell responses. Transfection with small interfering RNA (siRNA) reduced Zeb2 expression and resulted in decreased FcεRI-mediated degranulation, with a parallel reduction in receptor-induced activation of NFAT and NF-κB transcription factors, but an enhanced response to the LPS-mediated activation of NF-κB. There was variable and less of a decrease in the Ag-mediated release of the cytokines TNF-α, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple phosphorylation events were impaired that affected molecules both at early and late events in the signaling pathway. The Zeb2 siRNA-treated mast cells had altered cell cycle progression, as well as decreased expression of several molecules including cell surface FcεRI and its β subunit, Gab2, phospholipase-Cγ1, and phospholipase-Cγ2, all of which are required for receptor-induced signal transduction. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells.

Figure 1. Zeb2 regulates FcεRI-induced activation of NFAT and NF-kB transcription factors in mast cells

(A) MC9-NFAT-GFP or MC9-NF-kB-GFP cells were treated with individual and pooled siRNA specific for Zeb2 (indicated by 1 to 4 and P), or with control non-targeting siRNA. FcεRI- or LPS-induced activation of the transcription factors was determined by FACS analysis of GFP expression on day 1, 2 and 3 post-transfection. The results are expressed as a percentage of that in controls and are the average from 3 experiments. Cell lysates were analyzed by immunoblotting for expression of Zeb2 at day 1 (B), day 2 (C) and day 3 (D) in MC9-NFAT-GFP treated with control siRNA (lane labeled “C”) or individual and pooled siRNA’s for Zeb2 (lanes 1 to 4 and P). Anti-actin was used as loading control. Fraction of protein still expressed in cells is under each lane. Similar immunoblots were observed in 6 experiments (3 each for NFAT and NF-kB).

Figure 2. Zeb2 regulates FcεRI and ionophore-mediated cellular responses in primary mast cells

(A) Bone-marrow derived mouse mast cells (BMMC) were transfected with control non-targeting siRNA or the pool of four siRNA specific for Zeb2. The antigen-induced release of β-hexosaminidase, TNF-α IL-13 and CCL-4 were determined at day 1, 2 and 3 post-transfection (n=2). The changes in the cellular responses are presented as a percentage of that in control-transfected cells. (B) Cell lysates from BMMC transfected with non-targeting siRNA (“C”) or the Zeb2-specific (“T”) were analyzed by immunoblotting with antibodies to Zeb2 or actin. Fraction of protein still expressed in cells is under each lane. (C) Comparison of the IgE-antigen (Ag) and ionomycin/PMA responses in siRNA treated cells; changes in cellular responses are presented as a percentage of that in control transfected cells.

Figure 3. Cellular response of BMMC treated with single and pooled siRNA targeting Zeb2

(A) BMMC were treated with individual and pooled siRNA’s specific for Zeb2 (indicated by 1 to 4 and P), or with control non-targeting siRNA. Release of β-hexosaminidase, TNF-α IL-13 and CCL-4 was determined at day 1, 2 and 3 post-transfection. The results are expressed as a percentage of that in controls. Cell lysates were analyzed by immunoblotting for the expression of Zeb2 at day 1 (B), day 2 (C) and day 3 (D).

Figure 4. Knockdown of Zeb2 decreases FcεRI-induced phosphorylations

BMMC transfected with control scrambled siRNA or the single Zeb2-3 siRNA (“Treated”) were sensitized with IgE and then activated with antigen for the indicated times (minutes). Cell lysates were immunobloted with the anti-phospho-antibodies, then stripped and probed with the specific antibodies. Densitometry data were corrected for differences in loading using the actin blots and then compared to control cells treated with scrambled siRNA. In the case of PLCγ2 the change in the expression of this protein is also shown as there was decreased expression of this protein after treatment with siRNA specific for Zeb2. The different proteins as indicated are shown in (A) and (B). The results here are from day 3 (A) and day 2 (B) post-transfection. Similar results were in 3 different experiments and when the cells were treated with the pool of Zeb2 siRNA.

Figure 5. Knockdown of Zeb2 decreased cell-surface FcεRI expression

The expression of FcεRI was determined in BMMC treated with non-targeting siRNA or the single and the pooled siRNA specific for Zeb or Syk. The change in FcεRI expression was calculated from the Mean Fluorescence Intensity as determined by FACS (n=4) using the non-targeting siRNA transfected cells as controls.

Figure 6. Decreased Zeb2 expression alters cell cycle progression

BMMC were transfected with non-targeting siRNA (“Control”) or the most efficient single siRNA (Zeb2-3) and the pooled siRNA specific for Zeb2 (“Target”). Changes in the cell cycle were monitored by FACS. Percent of cells in G1 phase (A), S phase (B) and G2/M phase (C) at day 1, 2 and 3 post-transfection is presented (n=4).

Figure 7. Zeb2 regulates the expression of several proteins in mast cells

BMMC were treated with non-targeting siRNA (“Control”) or the single siRNA specific for Zeb2 (Zeb2-3) and the pooled siRNA’s (Zeb2-P). (A) Lysates from day 3 post-transfection were analyzed by immunoblotting with the indicated antibodies. Similar results were in 4–7 different experiments. (B) Densitometry data of the changes induced by decreased expression of Zeb2 as a percentage of controls; data is with both Zeb2-3 and Zeb2-pool siRNA form 6 to 15 determinations (X ± S.D.)

Figure 8. Zeb2 regulation of mast cell responses

The decrease in the expression of Zeb2 causes reduction in the level of other molecules in mast cells. Some of these are known to be part of the FcεRI signaling network. The decrease in cell surface FcεRI, together with reduced amounts of other signaling proteins results in decreased degranulation and cytokine release.