Abstract
Mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. A basic-helix-loop-helix leucine zipper transcription factor termed MITF is essential for the development of mast cells. Although other substances also linked to inflammatory reactions (such as various proteases and serotonin) require MITF for their expression, the role of MITF in eicosanoid generation has not been studied. We examined eicosanoid generation in bone marrow-derived mast cells (BMMCs) of tg/tg mice that lack MITF. Most eicosanoids generated by BMMCs are either prostaglandin (PG) D2 or leukotriene C4. The former is synthesized via the cyclooxygenase pathway, whereas the latter is synthesized via the 5-lipoxygenase pathway. In response to stimulation with IgE and antigens, BMMCs of tg/tg mice synthesized leukotriene C4 normally. However, neither immediate nor delayed PGD2 production was detected in these BMMCs. This indicates that MITF is a transcription factor that specifically activates the cyclooxygenase pathway, but not the 5-lipoxygenase pathway. Significant decreases in expression of hematopoietic PGD2 synthase (hPGDS, a terminal synthase for PGD2) were observed at both mRNA and protein levels in tg/tg BMMCs. MITF transactivated the hPGDS gene via a CACCTG motif located in the promoter region. MITF appeared to be essential for generation of PGD2 by enhancing expression of the hPGDS gene in BMMCs.
Highlights
Stimulation of bone marrow-derived mast cells (BMMCs) by cross-linking of Fc⑀RI with IgE and antigens elicits secretory granule exocytosis and immediate eicosanoid generation [15, 16]
When BMMCs are cultured in a medium containing IL-10, IL-1, and Kit ligand (KitL), the amount of generated prostaglandin D2 (PGD2) increases further up to 8 h after stimulation
These immediate and delayed phases of PGD2 generation were not observed in BMMCs derived from tg/tg mice, indicating that MITF is essential for both phases of PGD2 generation
Summary
Mice and Cells—The original stock of tg/tg mice, in which the mouse vasopressin-Escherichia coli -galactosidase transgene was integrated at the promoter region of the MITF gene, was provided by Dr H. The amount of PGD2 generated was measured in BMMCs preincubated with 1 g/ml indomethacin (Sigma) In these cases, BMMCs were sensitized with anti-DNP IgE in the presence of indomethacin, washed, and elicited with DNP-HSA. Luciferase Assay—The DNA fragment containing the promoter and 5Ј-untranslated region of the hPGDS gene (nt Ϫ1500 to ϩ200, where ϩ1 is a transcription initiation site) was amplified by PCR from genomic DNA of ϩ/ϩ BMMCs with LA-Taq DNA polymerase (Takara, Kyoto, Japan). This fragment was cloned upstream of the luciferase gene, and the reporter plasmid was constructed. Polyacrylamide gels were dried on Whatman 3MM chromatography paper and subjected to autoradiography
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
