Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcepsilonRI-activated RBL-2H3 basophilic cells

Abstract

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcepsilonRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.

Fig. 1

Species specificity and sequence of each siRNA against Syk. The sequence of the Syk siRNAs A–J is shown along with the specific of location homology to the rat Syk mRNA (RefSeq_dna: NM_012758). The species specificity indicates that this siRNA has 100% homology with the given mRNA for either rat, mouse or human Syk. CDS; coding sequence, 3′UTR; 3′ untranslated region, bp; base pair number.

Fig. 2

Identification of active siRNAs against rat Syk. (A) RBL-2H3 cells were transfected with Syk siRNAs A-J. In addition, three controls were used. (1) untreated cells, (2) cells transfected with GAPDH siRNA, (3) cells electroporated in the absence of siRNAs. Syk mRNA levels were measured 2 days after transfection by TaqMan PCR. Expression of Syk was normalized to levels of RNA polymerase II mRNA and then expressed as the percentage expression compared to that in untreated cells. Error bars represent the standard deviation of the mean for triplicate determinations from three separate experiments. A paired Student’s t-test was used to compare the statistical significance of the difference between data points. With the exception of Syk siRNA D, each Syk siRNA significantly reduced Syk mRNA expression relative to the GAPDH siRNA control with a p-value of less than 0.05. The percentage knockdown of Syk mRNA compared to untreated cells is also presented. (B) Total cell lysates were analyzed for levels of Syk protein by Western blot. Levels of β-actin were examined to control for protein loading. Note that two Syk proteins (Syk and the smaller SykB) are expressed in RBL-2H3 cells as a result of alternate mRNA splicing .

Fig. 3

Expression of IFIT1 in response to siRNA transfection. MEF cells were transfected with Syk siRNAs A, B, C, G, H, I and J and the control siRNA against GAPDH and the expression of IFIT1 determined 2 days later using TaqMan PCR. IFIT1 expression was normalized to levels of RNA polymerase II mRNA and then expressed as the percentage expression compared to that in cell treated with the transfection reagent alone. Error bars represent the standard deviation of the mean for duplicate determinations from two separate experiments. A paired Student’s t-test was used to compare the statistical significance of the difference between data points. With the exception of Syk siRNAs C and H (marked with an asterisk), no significant increase in IFIT1 expression relative to the GAPDH siRNA control was observed with a p-value of less than 0.05.

Fig. 4

Knockdown of Syk reduces FcεRI-mediated signal transduction in a manner similar to Syk kinase inhibitors. (A) RBL-2H3 cells were transfected with active Syk siRNAs A, G and J or the control siRNA against GAPDH and then treated with or without the FcεRI-crosslinking antibody. Total cell lysates were analyzed by Western blot for expression and phosphorylation of Syk (tyrosine 346), SLP-76 (tyrosine 128), LAT (tyrosine 226), PLCγ1 (tyrosine 783), Erk (threonine 202/tyrosine 204) and total tyrosine-phosphorylated proteins. Total levels of each protein as well as β-actin were also analyzed. (B) Untransfected RBL-2H3 cells were treated with the Syk small molecule kinase inhibitors BAY61-3606 (1 and 0.1 μM), R406 (1 and 0.1 μM) and piceatannol (20, 10 and 1 μM) and then activated with the FcεRI-crosslinking antibody. The phosphorylation of the same proteins mentioned in part A of this figure was analyzed by Western blot. Total Syk and β-actin levels were analyzed to control for protein loading.

Fig. 5

Syk siRNAs and inhibitors block FcεRI-mediated β-hexosaminidase release. RBL-2H3 cells were treated with Syk siRNAs (A) and kinase inhibitors (B) and then activated by overnight incubation in the mouse anti-DNP IgE antibody followed by addition of DNP-BSA. β-Hexosaminidase activity was measure in culture supernatants as described in Section 2. Error bars represent the standard deviation of the mean for quadruplicate determinations. A paired Student’s t-test was used to compare the statistical significance of the difference between data points. All treatments with Syk siRNAs and inhibitors led to a significantly reduced secretion of β-hexosaminidase compared to the control with a p-value of less than 0.05.