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Abstract: The CCAAT/enhancer-binding protein α (C/EBPα) is the member of a family of related basic leucine zipper (bZIP) transcription factors and is critical for granulopoiesis. We previously demonstrated that C/EBPα interacts with the ETS domain of widely expressed GABPα, which leads to cooperative transcriptional activation of the myeloid-specific promoter for human FCAR encoding the Fc receptor for IgA (FcαR, CD89) in part by facilitating recruitment of C/EBPα to the promoter. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. We demonstrate here that GABPα interacts with the last 18 residues of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerizing region. Deletion of this C-terminus resulted in loss of GABPα interaction but not affecting its DNA binding ability, indicating that it is not required for homodimer formation. Moreover, the C-terminus confers the ability to functionally synergize with GABP on a heterologous TAD when fused to the C-terminus of the VP16 TAD. We identified a three-amino acid stretch (amino acids 341–343) that is important for both functional and protein interactions with GABP. Ectopic expression in K562 cells of C/EBPα mutant incapable of interacting with GABPα does not induce expression of granulocytic differentiation markers including CD15, CD11b, GCSF-R and C/EBPε, and does not inhibit proliferation, whereas wild type does. These results demonstrate the functional importance of the C/EBPα C-terminus beyond the bZIP DNA-binding and dimerization region, which may mediate cooperative activation by C/EBPα and GABP of myeloid-specific genes involved in C/EBPα-dependent granulopoiesis. [Copyright &y& Elsevier]

More than 10 splice variants of the Fc receptor for IgA (FcαR, CD89) have been identified in human myeloid cells. In this study, we quantified FcαR splice transcripts ΔEC2 and Δ66EC2, which lack the entire and a part of the homologous immunoglobulin-like extracellular domain 2 (EC2), respectively. Tumor necrosis factor-α was found to specifically increase the ratio of ΔEC2 to the wild type CD89 in neutrophils and conversely decrease the ΔEC2 ratio in monocytes. We also observed a significant decrease in the neutrophil ΔEC2/CD89 ratio in pneumonia patients. These results suggest that ΔEC2 is differentially regulated and could be involved in immunoregulation of IgA-mediated host defense. [Copyright &y& Elsevier]

Abstract: The high affinity IgE Fc receptor (FcεRI) β chain is well implicated as a signal amplifier through the immunoreceptor tyrosine-based activation motif (ITAM) in its C-terminal intracellular region. Our previous study, however, demonstrated that mutation in all of the three tyrosine residues within the FcεRIβ ITAM did not impair FcεRI-induced cytokine production, suggesting a possible functional region other than the ITAM. To investigate the ITAM-independent mechanism by which FcεRIβ regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIβ mutants were generated. We observed that truncation of the FcεRIβ C-terminus downstream of the ITAM resulted in a considerable decrease in FcεRI-induced IL-6 production but not degranulation. Furthermore, mutagenesis of a single C-terminal aspartic acid (D234) to alanine (β-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and β-D234A suggests that the secondary structure of the FcεRIβ C-terminus was not affected by the D234A mutation. Consistently, we did not observe any effect of this mutation on FcεRI-induced tyrosine phosphorylation of FcεRIβ. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIβ ITAM. [Copyright &y& Elsevier]

Abstract: Besides mast cells and basophils, the high-affinity IgE Fc receptor (FcɛRI) is exclusively expressed on certain FcαR (IgA Fc receptor)-expressing immune cells such as neutrophils in allergic patients. Transfected rat basophilic leukemia cell line (RBL-2H3) co-expressing FcɛRI and FcαR was analyzed for effects of simultaneous receptor engagement by their specific antibodies on degranulation and signaling. Whereas supraoptimal FcɛRI engagement decreased degranulation, which is known as a bell-shaped dose–response curve, such inhibitory effect was not observed with FcαR engagement. However, simultaneous engagement of FcɛRI and FcαR showed that supraoptimal FcɛRI engagement down-regulates FcαR-mediated degranulation. This inhibition was associated with extensive phosphorylation of inositol polyphosphate 5′-phosphatase SHIP1 and FcɛRIβ, and reversed by adding actin-depolymerizing drug, latrunculin B. The results suggest an endogenous mechanism by which FcαR functionality is down-regulated in an ‘allergic environment’ where FcɛRI is co-expressed and extensively cross-linked on FcαR-expressing effector cells. [Copyright &y& Elsevier]

The Fc receptor γ-chain (FcRγ), which was first identified as a constituent of the high affinity IgE receptor, associates with various cell surface receptors to mediate intracellular signals. We identified three transcriptional enhancer elements in the 5′ region of the human FcRγ gene; one of the cis-elements was recognized by the transcription factor Sp-1 and another was recognized by GABP or Elf-1. The sequence of the other element was similar to a binding motif of the C/EBP family. Overexpression experiments showed that these transcription factors cooperatively activated the FcRγ promoter. Furthermore, inactivation of the GABP-binding site by nucleotide substitutions as well as repression of GABPα expression by RNA interference reduced Sp1-mediated transactivation of the FcRγ promoter, demonstrating that Sp1 and GABP synergistically activated the FcRγ promoter. This synergistic activation was suggested to require physical interaction between the two transcription factors, because the Ets domain of GABPγ was demonstrated to directly bind Sp1. On the other hand, GABP and Elf-1, whose recognition sequences overlapped, were shown to bind the FcRγ gene with similar affinity in the context of chromatin, although Elf-1 exerted weaker enhancer activity for FcRγ gene expression than did GABP. Both were thought to compete for binding to the element, because additional expression of Elf-1 in combination with Sp1 and GABP reduced FcRγ promoter activity. Such functional and physical interactions among transcription factors involved in the cooperative regulation of FcRγ gene expression as revealed in this study will become promising targets for medical applications against various immune diseases involving FcRγ. [ABSTRACT FROM AUTHOR]