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.

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.

Development of pyrrole-imidazole polyamide targeting fc receptor common gamma chain for the treatment of immune-complex related renal disease.

Fcγ receptors I and III are thought to be involved in the development of lupus nephritis. Expression of Fc receptor common gamma chain (FcRγ) is necessary for the stable expression of Fcγ receptors I and III. The aim of this study was to develop a novel agent for the treatment of immune complex related renal disease using a gene regulator, pyrrole(Py)-imidazole(Im) (PI) polyamide, targeting the mouse FcRγ gene promoter. Two PI polyamides targeting FcRγ promoters were designed and synthesized. The effect of the PI polyamides on FcRγ mRNA expression was evaluated in J774.A cells by real-time polymerase chain reaction (PCR), and CD16/32 protein expression was determined by immunocytochemical analysis and flow cytometry. The effects of these polyamides on FcRγ gene expression and CD16/32 protein expression were evaluated in mouse peripheral blood mononuclear cells (PBMCs). One milligram per kilogram body weight of PI polyamide was injected via the tail vein every 2 d for 1 week and PBMCs were collected and analyzed. PI polyamide showed a specific binding to the target DNA in a gel mobility shift assay. Treatment of J774.A cells with 1.0 µM PI polyamide 1 significantly reduced FcRγ mRNA expression and CD16/32 surface protein expression in J774.A cells. Similarly, PI polyamide significantly decreased expression of FcRγ mRNA and CD16/32 in the PBMCs of C57B6 mice. PI polyamide designed to bind the FcRγ promoter decreased FcRγ gene and CD16/32 protein expression. PI polyamide targeting the FcRγ gene may be a novel gene regulator for the prevention of lupus nephritis or other immune complex-related disease.

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

The high affinity IgE Fc receptor (FcεRI) ß 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 FcεRIß 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 FcεRIß regulates FcεRI-induced cytokine production, mouse mast cells expressing various FcεRIß mutants were generated. We observed that truncation of the FcεRIß 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 (ß-D234A) also significantly impaired IL-6 production. In addition, the similarity between the circular dichroism (CD) spectra of the wild type and ß-D234A suggests that the secondary structure of the FcεRIß 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 FcεRIß. These observations strongly suggest a novel signaling pathway mediated by the cytoplasmic tail downstream of the FcεRIß ITAM.

Amino acid residues in the beta3 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.

High affinity receptor for IgE stimulation activates protein kinase D augmenting activator protein-1 activity for cytokine producing in mast cells.

Protein kinase D (PKD) is a serine-threonine kinase involved in the activation of a variety of cells. In mast cells, activation of PKD by cross-linking of high affinity receptor for IgE (FcepsilonRI) has been reported, but little is known for its effects on cytokine production. We investigated the roles of PKD on FcepsilonRI-induced activator protein-1 (AP-1) activation and proinflammatory cytokine productions in mast cells. Pharmacological inhibition of PKD strongly inhibited production of interleukin (IL)-13 and tumor necrosis factor (TNF)-alpha induced by FcepsilonRI stimulation, and the overexpression of PKD significantly increased the IL-13 and TNF-alpha production. Reporter assay revealed that the overexpression of PKD enhanced FcepsilonRI-induced IL-13 promoter activation, and that the 5'-flanking region of IL-13 gene from positions -110 to -52 was under the regulation of PKD. The overexpression of PKD enhanced the induction of AP-1 luciferase activity by FcepsilonRI stimulation, while it had no effect on luciferase activities dependent upon NF-kappaB and NF-AT activated by FcepsilonRI stimulation. In EMSA, c-Jun and c-Fos appear to be the major components of AP-1 complexes activated by FcepsilonRI stimulation. Moreover the overexpression of PKD strongly enhanced the phosphorylation of both c-Jun and c-Fos following FcepsilonRI stimulation. Although stress-activated protein kinase/c-Jun N-terminal kinase (JNK) is known to be an important regulator for c-Jun phosphorylation and AP-1 activation, overexpression and inhibition of PKD had no effects on JNK phosphorylation. These results suggest that PKD may play a pivotal role in FcepsilonRI-induced cytokine production in mast cells through the activation of c-Jun, c-Fos, and AP-1.

Functionality of the IgA Fc receptor (FcalphaR, CD89) is down-regulated by extensive engagement of FcepsilonRI.

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

Cooperative regulation of Fc receptor gamma-chain gene expression by multiple transcription factors, including Sp1, GABP, and Elf-1.

The Fc receptor gamma-chain (FcRgamma), 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 FcRgamma 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 FcRgamma promoter. Furthermore, inactivation of the GABP-binding site by nucleotide substitutions as well as repression of GABPalpha expression by RNA interference reduced Sp1-mediated transactivation of the FcRgamma promoter, demonstrating that Sp1 and GABP synergistically activated the FcRgamma promoter. This synergistic activation was suggested to require physical interaction between the two transcription factors, because the Ets domain of GABPalpha was demonstrated to directly bind Sp1. On the other hand, GABP and Elf-1, whose recognition sequences overlapped, were shown to bind the FcRgamma gene with similar affinity in the context of chromatin, although Elf-1 exerted weaker enhancer activity for FcRgamma 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 FcRgamma promoter activity. Such functional and physical interactions among transcription factors involved in the cooperative regulation of FcRgamma gene expression as revealed in this study will become promising targets for medical applications against various immune diseases involving FcRgamma.

Priming of peripheral monocytes with prolactin (PRL) sensitizes IFN-gamma-mediated indoleamine 2,3-dioxygenase (IDO) expression without affecting IFN-gamma signaling.

Prolactin (PRL) was originally identified by its ability to stimulate mammary development and lactation, and its essential roles other than lactation have recently been implicated in female reproduction. However, little is known about PRL-mediated events in pregnancy. The tryptophan catabolism enzyme indoleamine 2,3-dioxygenase (IDO) is interferon-gamma (IFN-gamma)-inducible and has recently become a focus for maternal-fetal tolerance for successful pregnancy. Based on recognition that PRL is one of the up-regulated hormones in pregnancy, in a previous study we have shown that PRL induces IDO expression in monocytes in cooperation with a suboptimal concentration of IFN-gamma. Here, we demonstrate that PRL sensitizes monocytes to induce IDO expression in response to low doses of IFN-gamma without affecting the typical IFN-gamma signaling events, such as STAT1 phosphorylation and IRF-1 induction. In addition, IDO induction in these cell cultures was observed only after 24 h pre-exposure to PRL. These results indicate a priming effect of PRL on monocytes that occurs before IFN-gamma signaling and increases their sensitivity to IFN-gamma for IDO induction, rather than a synergistic effect of PRL and IFN-gamma on IDO induction. These results offer new insights into the roles of PRL in female reproduction, as well as provide a better understanding as to how IDO expression is regulated and achieved in pregnancy.

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

Fc receptor for IgA (FcalphaR, 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 FcalphaR 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/microl) (5.1 and 8.5%) compared with 91 patients with normal PLT (> or = 150,000/microl) (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 FcalphaR expression affecting IgA-mediated immune responses and/or IgA catabolism.

C/EBPalpha functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (Fc alphaR, CD89) gene promoter.

Human Fcalpha receptor (Fc alphaR; CD89), the receptor for the crystallizable fragment (Fc) of immunoglobulin A (IgA), is expressed exclusively in myeloid cells, including granulocytes and monocytes/macrophages, and is considered to define a crucial role of these cells in immune and inflammatory responses. A 259-base pair fragment of the FCAR promoter is sufficient to direct myeloid expression of a reporter gene and contains functionally important binding sites for CCAAT/enhancer-binding protein alpha (C/EBPalpha) (CE1, CE2, and CE3) and an unidentified Ets-like nuclear protein. Here, we show that the Ets-binding site is bound by a heterodimer composed of GA-binding protein alpha (GABPalpha), an Ets-related factor, and GABPbeta, a Notch-related protein. Cotransfection of GABP increased FCAR promoter activity 3.7-fold through the Ets-binding site. GABP and C/EBPalpha synergistically activated the FCAR promoter 280-fold. Consistent with these observations, in vitro binding analyses revealed a physical interaction between the GABPalpha subunit and C/EBPalpha. This is the first report demonstrating both physical and functional interactions between GABP and C/EBPalpha and will provide new insights into the molecular basis of myeloid gene expression.

Alternative splicing of myeloid IgA Fc receptor (Fc alpha R, CD89) transcripts in inflammatory responses.

More than 10 splice variants of the Fc receptor for IgA (Fc alpha R, CD89) have been identified in human myeloid cells. In this study, we quantified Fc alpha R splice transcripts Delta EC2 and Delta 66 EC2, which lack the entire and a part of the homologous immunoglobulin-like extracellular domain 2 (EC2), respectively. Tumor necrosis factor-alpha was found to specifically increase the ratio of Delta EC2 to the wild type CD89 in neutrophils and conversely decrease the Delta EC2 ratio in monocytes. We also observed a significant decrease in the neutrophil Delta EC2/CD89 ratio in pneumonia patients. These results suggest that Delta EC2 is differentially regulated and could be involved in immunoregulation of IgA-mediated host defense.

C/EBP alpha and Ets protein family members regulate the human myeloid IgA Fc receptor (Fc alpha R, CD89) promoter.

Fc alpha R (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 Fc alpha R 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/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R 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 Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.

Transcriptional regulation of Fcgr2b gene by polymorphic promoter region and its contribution to humoral immune responses.

FcgammaRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcgammaRIIB1 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 FcgammaRIIB1 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 FcgammaRIIB1 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.