BMMCs with NTAL KD exhibit increased degranulation and calcium response.

<p>(A) IgE-sensitized BMMCs (WT, NTAL KO, NTAL KD, and WT pLKO) were stimulated for 30 minutes with various concentrations of Ag (TNP-BSA), and β-glucuronidase released into supernatant was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105539#s2" target="_blank">Materials and Methods</a>. Data represent means ± SE from 7–17 independent experiments performed in duplicates or triplicates). (B) IgE-sensitized BMMCs were stimulated with Ag [TNP-BSA at a concentration 100 ng/ml (Ag-100) or 500 ng/ml (Ag-500)], SCF (40 ng/ml), or both activators together. Data represent means ± SE from 6–20 independent experiments). (C–E) BMMCs were sensitized with IgE, loaded with Fura-2-AM (1 µg/ml), then stimulated with Ag (100 ng/ml TNP-BSA; C), SCF (40 ng/ml; D) or both activators together (E) and free intracellular Ca²⁺ was monitored by measuring fluorescence emission at 510 nm after excitation at 340 and 380 nm. Arrows indicate addition of Ag and/or SCF. Data are means ± SE from 11 (C), 7 (D) or 6 (E) independent experiments performed in duplicates. All data presented in A–E were obtained with BMMCs isolated from 3–5 mice. *^,+<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; in A, C and E, significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs and WT pLKOs (crosslets) are shown.</p

BMMCs with NTAL KD exhibit enhanced actin depolymerization after stimulation with Ag or Ag + SCF.

<p>Cells were activated with Ag (250 ng/ml TNP-BSA; A), SCF (40 ng/ml; B) or Ag + SCF (C). At the indicated times, the cells were fixed, stained for F-actin with Alexa Fluor 488-phalloidin and analyzed by flow cytometry. Data were normalized to fluorescence of resting cells (similar in all cell types). Values indicate mean ± SE (n = 6). *^,+<i>p</i><0.05; **^,++<i>p</i><0.01; ***^,+++<i>p</i><0.001; significant differences between NTAL KOs and WTs (asterisks) and NTAL KDs vs WT pLKOs (crosslets) are shown.</p

Multiple Regulatory Roles of the Mouse Transmembrane Adaptor Protein NTAL in Gene Transcription and Mast Cell Physiology

<div><p>Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.</p></div

Decreased NTAL expression after shRNA silencing.

<p>(A) BMMCs were infected with five lentiviral shRNA constructs (NTAL KD 1–5) or empty pLKO.1 construct (WT pLKO). After selection in puromycin, the amount of NTAL was assessed by immunoblotting. For comparison, NTAL in noninfected WT and NTAL KO cells was also evaluated. Actin was used as a loading control. (B) Densitometry analysis of NTAL immunoblots. The data were normalized to the amount of NTAL in WT pLKO cells and that of actin. Means ± SD were calculated from 3–7 independent experiments. ***<i>p</i><0.001.</p

Primers used in quantitative RT-PCR.

<p>Primers used in quantitative RT-PCR.</p

Principal component analysis of the microarrays.

<p>Each colored circle represents different cell type: NTAL KD (KD; red), NTAL KO (KO; blue), WT pLKO (pLKO; green), and WT (lilac). Each mouse from which the cells originated is identified by a colored numbers. The WT BMMCs isolated from mice 1–3 (blue numbers) were used not only as controls for NTAL KO cells, but also for obtaining NTAL KD and WT pLKO cells after lentiviral infection with NTAL shRNA or empty pLKO vector, respectively. The BMMC isolated from <i>Lat</i>^-/- mice 4–6 (lilac numbers) were used only as cells with NTAL KO. Treatment [Ag-activated cells (2h, pink) and nonactivated cells (0h, green)] is distinguished by ellipsoids. The arrays cluster according to the treatment groups showing separation along PC #1 and according to the type of cells showing separation along PC #2. The percentage values indicate the proportion of total variance described by each PC; PC #1 (X-axis), PC #2 (Y-axis), and PC #3 (Z-axis).</p

NTAL-dependent up- or down-regulated gene transcripts in resting and Ag-activated BMMCs.

<p>(A) Venn diagram illustrating number of genes with expression significantly (>1.8 x) up- or down-regulated in nonactivated cells with NTAL KO vs WT cells and NTAL KD vs WT pLKO cells; only 9 genes showed overlap. (B) Fold up- or down-regulation of overlapping genes in cells with NTAL KO and NTAL KD as determined by microarray data and qPCR analysis. Not shown are genes where qPCR data were inconsistent (IC) with microarray data and data with no significant p-values in microarray data analysis (NS). Four down-regulated genes involved in cholesterol synthesis in NTAL KO cells are also included (<i>Idi1, Fdps, Lss</i> and <i>Pmvk</i>). (C) Venn diagram as in A but documenting genes in Ag-activated NTAL KO and KD cells; only 5 genes showed overlap. (D) Microarray data and qPCR analysis of the overlapping genes in Ag-activated NTAL KO and KD cells as described in C; only 4 genes showing transcriptional regulation in the same direction are shown.</p

FcεRI-activated BMMCs with NTAL KD show enhanced tyrosine phosphorylation of ERK and LAT.

<p>NTAL-deficient cells and WT controls were sensitized with IgE and stimulated with Ag (100 ng/ml TNP-BSA). After the indicated time intervals, the cells were solubilized in lysis buffer and postnuclear supernatants were analyzed by immunoblotting for tyrosine phosphorylated ERK (pERK; A) or LAT (pLAT; B), followed by stripping and immunoblotting for total ERK (A) or LAT (B). Fold inductions of protein tyrosine phosphorylation, normalized to nonactivated WT cells and corrected for the amount of protein in each lysate, are also included. Each immunoblot is a typical result from three independent experiments.</p

A hypothetical model on the role of NTAL in mast cell activation and transcriptional regulation.

<p>(A) In nonactivated WT cells both adaptor proteins, LAT and NTAL, and FcεRI β and γ subunits only exhibit weak phosphorylation, low [Ca²⁺]_i and transcription corresponding to nonactivated cells (Transcription profile 1). (B) After Ag-mediated aggregation of the FcεRI-IgE complex, β and γ subunits of the FcεRI are tyrosine phosphorylated by LYN and SYK. SYK then phosphorylates NTAL and LAT and this leads to enhanced Ca²⁺ uptake and further propagation of the signal, including dramatic changes in transcriptional regulation (Transcription profile 2). (C) In nonactivated NTAL-deficient cells, LAT and FcεRI subunits are only weakly tyrosine phosphorylated and the cells exhibit slightly different transcriptional regulation when compared to WT cells (Transcription profile 3). (D) After FcεRI triggering of NTAL-deficient cells, β and γ subunits of the FcεRI are tyrosine phosphorylated as in B, but because of the absence of NTAL, LAT is more phosphorylated by SYK. This leads to enhanced mobilization of Ca²⁺ and other signaling events and transcriptional regulation which differs from the one in activated WT cells (Transcription profile 4).</p

Differences in transcriptional regulation between NTAL KO and WT cells.

<p>Differences in transcriptional regulation between NTAL KO and WT cells.</p