Toshibumi Shimokawa

Transcriptional Regulation of Fcgr2b Gene by Polymorphic Promoter Region and Its Contribution to Humoral Immune Responses

FcγRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcγRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcγRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position −302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcγRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.

Cooperative Regulation of Fc Receptor γ-Chain Gene Expression by Multiple Transcription Factors, Including Sp1, GABP, and Elf-1

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 GABPα 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.

C/EBPα and Ets Protein Family Members Regulate the Human Myeloid IgA Fc Receptor (FcαR, CD89) Promoter

FcαR (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the FcαR 5′-flanking region from −102 to −64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at −74 and −92, respectively. EMSAs and cotransfection experiments show that C/EBPα acts as a major activator of the FcαR promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBPα at −139 and −127. On the other hand, the FcαR Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces FcαR promoter activity, suggesting some functional interaction between these factors. As the in vivo role of FcαR is still incompletely defined, these findings reveal the features controlling the FcαR promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of FcαR gene expression associated with its potential roles.

Cooperative regulation of FcRγ gene expression by multiple transcription factors including Sp1, GABP and Elf-1

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 GABPα 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.

Highly expressed cytoplasmic FcεRIβ in human mast cells functions as a negative regulator of the FcRγ‐mediated cell activation signal

We recently reported that the interaction between Lyn and FcεRIβ is indispensable for FcεRI‐mediated human mast cell (MC) activation and that FcεRIβ functions as an amplifier of FcεRI‐mediated activation signal. Some of FcεRIβ in cytoplasm appeared not to be co‐localized with FcεRIα. The function of FcεRIβ in the cytoplasm remains unknown.

Regulation of nephrin gene by the Ets transcription factor, GA-binding protein

BACKGROUND Transcription factor GA-binding protein (GABP) is suggested to be involved in the formation of the neuromuscular junctions by regulating the transcription of synapse genes. Interestingly, neurons and podocytes share molecular and functional similarities that led us to investigate the expression and function of GABP in podocytes and its role in transcriptional regulation of nephrin, the key molecule of the podocyte slit diaphragm that is essential for normal glomerular ultrafiltration. METHODS The expression and localization of GABP in the rat and human kidney as well as in human embryonic kidney A293 cells and undifferentiated and differentiated human podocytes were analysed by immunoblotting and immunostaining. The role of GABP in activating the nephrin promoter was investigated by reporter gene assay and site-directed mutagenesis of the GABP-binding elements, and the interaction of GABP with the nephrin promoter was analysed by chromatin immunoprecipitation. The function of GABP in podocytes was studied by knocking down GABPα in differentiated human podocytes using lentiviral shRNA targeting GABPα. RESULTS GABP is expressed in the nuclei in rat and human glomeruli. In addition, in A293 cells and undifferentiated and differentiated human podocytes, GABP highly enriches in the nucleus. GABP activates and binds nephrin proximal promoter and Ets sites are essential for this activity. Knock-down of GABPα stimulates apoptosis in cultured podocytes. CONCLUSIONS The results show that GABP is expressed in podocytes and is involved in the regulation of nephrin gene expression. Furthermore, GABP may be important in the maintenance of podocyte function by regulating apoptosis.

Genetic variants of the IgA Fc receptor (FcαR, CD89) promoter in chronic hepatitis C patients

Fc receptor for IgA (FcαR, CD89) is capable of triggering IgA-mediated immune responses to pathogens and has been proposed to function in circulating IgA clearance. Because inheritable variations modifying individual immune responses or immunoglobulin catabolism may affect the chronicity of viral infection, we investigated whether promoter polymorphisms of the FcαR gene (FCAR) affect chronic hepatitis C virus (HCV) infection and its disease progression. The two −311T/C and −142T/C single-nucleotide polymorphisms (SNPs) were studied by direct DNA sequencing in 177 Japanese patients with chronic hepatitis C (CHC). Both −311CC and −142CC genotypes were more frequent in CHC patients (15.9 and 18.6%) compared with 210 healthy controls (5.7 and 10.0%) [p = 0.001, odds ratio (OR) = 3.10, 95% confidence interval CI) = 1.53–6.30 and p = 0.014, OR = 2.06, 95% CI = 1.14–3.72, respectively], and were associated with infection with HCV genotype 2a/2b (p = 0.019 and p = 0.005, respectively). Conversely, −311CC and −142CC were decreased in 59 patients at advanced stages of disease as assessed on the basis of hepatic fibrosis markers such as decreased platelet count (PLT) ( < 150,000/μl) (5.1 and 8.5%) compared with 91 patients with normal PLT ( ≥ 150,000/μl) (24.2 and 26.4%) (p = 0.006 and p = 0.005, respectively). Moreover, among the patients with normal PLT (but not with decreased PLT), −311CC or −142CC was significantly associated with decreased serum IgA levels (p = 0.023 or p = 0.007, respectively). These results suggest that the FCAR promoter SNPs may be related to chronic HCV infection and disease progression in Japanese CHC, which might be explained by altered FcαR expression affecting IgA-mediated immune responses and/or IgA catabolism.

Identification of the C/EBPα C-terminal tail residues involved in the protein interaction with GABP and their potency in myeloid differentiation of K562 cells.

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.

FcεRI-induced mast cell cytokine production critically involves an aspartic acid residue (D234) in the C-terminal intracellular domain of the FcεRIβ chain.

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.

Amino acid residues in the β3 strand and subsequent loop of the conserved ETS domain that mediate basic leucine zipper (bZIP) recruitment and potentially distinguish functional attributes of Ets proteins

Ets family members share a conserved DNA-binding ETS domain, and serve a variety of roles in development, differentiation and oncogenesis. Besides DNA binding, the ETS domain also participates in protein-protein interactions with other structurally unrelated transcription factors. Although this mechanism appears to confer tissue- or development stage-specific functions on individual Ets proteins, the biological significance of many of these interactions remains to be evaluated, because their molecular basis has been elusive. We previously demonstrated a direct interaction between the ETS domain of the widely expressed GABPalpha (GA-binding protein alpha) and the granulocyte inducer C/EBPalpha (CCAAT/enhancer-binding protein alpha), and suggested its involvement in co-operative transcriptional activation of myeloid-specific genes, such as human FCAR encoding FcalphaR [Fc receptor for IgA (CD89)]. By deletion analysis, we identified helix alpha3 and the beta3/beta4 region as the C/EBPalpha-interacting region. Domain-swapping of individual sub-domains with those of other Ets proteins allowed us to highlight beta-strand 3 and the subsequent loop, which when exchanged by those of Elf-1 (E74-like factor 1) reduced the ability to recruit C/EBPalpha. Further analysis identified a four-amino acid swap mutation of this region (I387L/C388A/K393Q/F395L) that reduces both physical interaction and co-operative transcriptional activation with C/EBPalpha without affecting its transactivation capacity by itself. Moreover, re-ChIP (re-chromatin immunoprecipitation) analysis demonstrated that GABPalpha recruits C/EBPalpha to the FCAR promoter, depending on these residues. The identified amino acid residues could confer the specificity of the action on the Ets proteins in diverse biological processes through mediating the recruitment of its partner factor.