Gene expression profiling of Ca²⁺-ATPase inhibitor DTBHQ and antigen-stimulated RBL-2H3 mast cells

Abstract.

Objective and Design: Ca²⁺ signaling is critical for mast cell activation by antigen stimulation, and we previously described that the signaling can be mimicked by Ca²⁺-ATPase inhibitors. We therefore investigated the effect of the Ca²⁺-ATPase inhibitor and antigen stimulation on the gene expression profiles of RBL-2H3 mast cells.¶Material: A Ca²⁺-ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), an antigen (dinitrophenylated BSA), a high-density oligonucleotide microarray (Affymetrix GeneChip) technique, and a well-characterized rat mast cell line RBL-2H3 were used.¶Treatment: RBL-2H3 cells were activated for 3 h with 10 μM DTBHQ, which increases cytosolic Ca²⁺ concentration, or 10 μmg/ml antigen, which cross-links IgE receptors, and the mRNA expression profiles (8,799 genes) were analyzed with GeneChip arrays (n = 3).¶Methods: Expression levels were measured by GeneChip, and the differences were tested by Welch's t-test and P-values less than 0.05 were considered statistically significant. Values are expressed as means ±SEM.¶Results: The genes, including MCP-1, GADD45, Relaxin H1, CSF-1, c-jun-oncogene, Pyk-2, NKR-P2 and CREM, were significantly up-regulated by both DTBHQ and antigen stimuli, whereas the genes including interleukin (IL)-3, IL-4, IL-9, IL-13, GADD153, butyrate response factor, and Fas ligand, were up-regulated by DTBHQ alone. On the other hand, the expression of several genes, including GATA-1, were down-regulated by DTBHQ stimulation.¶Conclusions: These results suggest 1) that DTBHQ seems to induce proinflammatory responses by stimulating the production of several cytokines through the expression of several transcription factors, 2) that the changes in gene expression profile induced by DTBHQ and by IgE receptor cross-linking in mast cells were almost the same, but many more stress-inducible genes like GADD153 were up-regulated by the former.