[Show abstract][Hide abstract] ABSTRACT: It is well established that TGF-β1 and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-β1, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules α4β7 and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.
[Show abstract][Hide abstract] ABSTRACT: Ginsenosides are the major components of ginseng, which is known to modulate blood pressure, metabolism, and immune function, and has been used to treat various diseases. It has been reported that ginseng and several ginsenosides have immunoregulatory effects on the innate and T cell-mediated immune response. However, their effects on the humoral immune response have not been fully explored. The present study examined the direct effects of red ginseng extract (RGE) and ginsenosides on mouse B cell proliferation and on antibody production and the expression of germline transcripts (GLT) by mouse B cells in vitro. RGE slightly reduced B cell proliferation, but increased IgA production by LPS-stimulated B cells. Furthermore, ginsenoside Rg1 and 20(S)-Rg3 selectively induced IgA production and expression of GLTα transcripts by LPS-stimulated B cells. Collectively, these results suggest that ginsenoside Rg1 and 20(S)-Rg3 can drive the differentiation of B cells into IgA-producing cells through the selective induction of GLTα expression.
[Show abstract][Hide abstract] ABSTRACT: TGF-β induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-β signal-transducing transcription factors, mediate germline (GL) α transcription induced by TGF-β1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-β-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-β1-induced, Smad3/4-mediated GLα transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-β1-induced, Smad3/4-mediated GLα promoter activity, expression of endogenous GLα transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-β1-induced, Smad3/4-mediated GLα promoter activity. We found that PIASy overexpression suppresses the GLα promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLα transcription and IgA switching induced by TGF-β1/Smad3/4, while PIASy acts as a repressor.
[Show abstract][Hide abstract] ABSTRACT: Retinoic acid (RA) is known to have several functions that lead to a potent mucosal IgA response. Nevertheless, its exact role in human IgA synthesis has yet to be elucidated. Thus, we investigated the role of RA in promoting IgA isotype switching in human B cells. We found that RA increased IgA production and the expression of germ-line IgA1 and IgA2 transcripts (GLTα1 and GLTα2). This induction occurred alongside an increase in the frequency of IgA1-secreting B cell clones, as assessed by limiting dilution analysis. Under the same conditions, RA did not increase IgM and IgG production. Am80, an agonist of RA receptor α (RARα), increased IgA production. In addition, RA activity was abrogated by LE540, an antagonist of RAR, suggesting that the RAR pathway is involved in RA-induced IgA production. Taken together, these results indicate that RA induces IgA isotype switching mainly through RARα in human B cells.
No preview · Article · Jun 2014 · Human Immunology
[Show abstract][Hide abstract] ABSTRACT: Dectin-1, which specifically recognizes β-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, β-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.
[Show abstract][Hide abstract] ABSTRACT: The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF-β1. RA independently caused only IgA switching, whereas TGF-β1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA-secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF-β1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4β7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF-β have important effects on the overall gut IgA antibody response.
No preview · Article · Jun 2013 · Journal of leukocyte biology
[Show abstract][Hide abstract] ABSTRACT: Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.
[Show abstract][Hide abstract] ABSTRACT: Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cells. APRIL substantially increased both HoxC4 and AID expression, whereas BAFF induced the expression of AID but not HoxC4. To elucidate the underlying mechanisms, we constructed a HoxC4 gene promoter reporter vector and analyzed the promoter induction after APRIL stimulation. APRIL enhanced the HoxC4 promoter activity by 2.3-fold, and this increase disappeared when the second putative NF-κB-binding promoter element (NBE2) was mutated. Based on ChIP assays, we found that NF-κB bound to the HoxC4 promoter NBE2 region. Furthermore, the overexpression of NF-κB augmented the APRIL-induced HoxC4 promoter activity, while the expression of dominant negative-IκBα suppressed it. Taken together, our findings suggest that NF-κB mediates APRIL-induced HoxC4 transcription.
[Show abstract][Hide abstract] ABSTRACT: Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-β1-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-β1-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-β1-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-β1-nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.
[Show abstract][Hide abstract] ABSTRACT: The inhibitory effect of polyphenol extracts (Seapolynol(™), SPN) of the marine brown algae Ecklonia cava and dieckol, a major component of SPN, on hyperlipidemia was investigated in ICR mice fed a high-fat diet (HFD) for five weeks. For analysis of the anti-hyperlipidemic effects of SPN and dieckol, these two agents were given orally on a daily basis to HFD-fed mice for four weeks, starting one week after the beginning of HFD feeding. Groups administered with SPN as well as dieckol showed lower body weight gains than the HFD only group. Administration of SPN and dieckol also resulted in a significant reduction of the level of total cholesterol (TCHO), triglyceride (TG), and low-density lipoprotein (LDL) cholesterol in the serum of HFD-fed mice. In Oil Red O staining using 3T3-L1 preadipocytes, it was shown that both SPN and dieckol markedly inhibited lipid accumulation of 3T3-L1 cells. Furthermore, SPN and dieckol (50 μg/mL) significantly inhibited 3-hydroxyl-methyl glutaryl coenzyme A (HMGCoA) reductase activity in vitro. Taken together, these results suggest that polyphenols of Ecklonia cava (SPN) and dieckol reduce body weight gain and fat accumulation in HFD-induced obese mice, and that their hypolipidemic effect is related to the inhibition of adipogenesis of adipocytes and HMGCoA reductase activity.
Full-text · Article · Mar 2012 · Preventive Nutrition and Food Science
[Show abstract][Hide abstract] ABSTRACT: Retinoic acid (RA) is considered to possess an activity of IgA isotype switching. Thus far, TGF-β1 is known to be the most
powerful IgA isotype switch factor. To elucidate the molecular mechanisms underlying the Ig germ line (GL) α transcriptional
regulation by RA, we constructed three different sizes of mouse GLα promoter reporters; short-GLα(−130/+14), middle-GLα(−448/+72)
and long-GLα(−3028/+72). Based on luciferase assay, RA increased the activity of all three GLα promoter reporters by approximately
2-fold and the effect was further enhanced by TGF-β1. Overexpression of Smad3/4 increased TGF-β1-induced GLα promoter activities
but had no effect on RA-induced GLα promoter activities. In order to analyze the characteristics of the RA-inducible GLα promoter
region, we also constructed two mutant reporters: Smad3 binding elements (SBEs)-substituted short-GLα (short-GLα mSBE) and
Runx3 binding elements (RBEs)-substituted short-GLα (short-GLα mRBE) promoter reporters. Promoter activities of the two mutant
reporters to RA were comparable to that of wild type reporter, while those of the two mutant reporters to TGF-β1 were markedly
diminished as compared to that of WT short-GLα. Finally, RA-induced GLα transcription was virtually disappeared by LE540,
an antagonist of RA receptor (RAR). Taken together, these results suggest that RA induces GLα transcription mainly through
RAR pathway, where neither Smad3/4 nor Runx3 is involved.
KeywordsRetinoic acid–TGF-β1–GLα promoter–IgA–Smad–RA receptor
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a multi-step process that involves the activation, proliferation, and migration of endothelial cells. We have recently shown that TGF-beta1 can induce mouse macrophages to produce VEGF, a potent angiogenic factor. In the present study, we explored whether TGF-beta1 has a similar effect on mouse dendritic cells. First, we show that under hypoxic conditions, TGF-beta1 induced the expression of VEGF transcripts in bone marrow-derived dendritic cells. Overexpression of Smad3/4 further augmented TGF-beta1-induced VEGF transcription, while overexpression of DN-Smad3 decreased VEGF transcription in DC2.4 cells, a mouse dendritic cell line. We also show that TGF-beta1 and Smads are involved in the induction of VEGF protein secretion. Interestingly, under the same conditions, the expression of VEGF receptor 1 (Flt-1) was also elevated at both the transcriptional and protein levels. Additionally, we found that the TGF-beta1-induced VEGF secretion in activated DC2.4 cells has wound-healing properties. Finally, Smad7 and Smurf1 negatively regulated the TGF-beta1-induced and Smad3/4-mediated VEGF expression. Taken together, these results indicate that TGF-beta1 can enhance the expression of VEGF and Flt-1 through the typical Smad pathway in mouse dendritic cells.
Preview · Article · Sep 2010 · Experimental and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key protein in CSR, targets immunoglobulin H (IgH) switch regions, which contain 5'-AGCT-3' repeats in their core. How AID is recruited to switch regions remains unclear. Here we show that 14-3-3 adaptor proteins have an important role in CSR. 14-3-3 proteins specifically bound 5'-AGCT-3' repeats, were upregulated in B cells undergoing CSR and were recruited with AID to the switch regions that are involved in CSR events (Smu-->Sgamma1, Smu-->Sgamma3 or Smu-->Salpha). Moreover, blocking 14-3-3 by difopein, 14-3-3gamma deficiency or expression of a dominant-negative 14-3-3sigma mutant impaired recruitment of AID to switch regions and decreased CSR. Finally, 14-3-3 proteins interacted directly with AID and enhanced AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.
[Show abstract][Hide abstract] ABSTRACT: p300 acts as a coactivator for TGF-β1-induced Ig germ-line (GL) α transcription. To understand the exact role of p300, we
analyzed known domains of p300 using several deletion mutants. p300 mutants lacking both the histone acetyltransferase (HAT)
and Smad-binding domains did not enhance GLα promoter activity. In addition, Smad-binding domain within p300 was not sufficient
for the promoter activity. Surprisingly, overexpression of a p300 mutant retaining the HAT domain but lacking the rest of
downstream segment, p300[N1736], greatly augmented the GLα promoter activity, regardless of TGF-β1 treatment, to levels much
higher than that of p300[WT]. Together, these results suggest that HAT activity of p300 is required for GLα promoter activity.
We found that p300[N1736] binds to the GLα promoter using ChIP assays. Further analysis of p300[N1736] indicated that both
Stat1 and HDAC1 can inhibit the enhancing effect of p300[N1736], suggesting the importance of the Stat1-binding domain and
the HAT domain. E1A, a repressor of p300[WT], had no effect on the activity of p300[N1736], which also lacks the E1A-binding
domain. Finally, when the amount of transfected p300[N1736] was reduced 40-fold, p300[N1736] functioned similarly to overexpressed
p300[WT]. Thus, p300 [N1736], though not having a Smad3-binding domain, augmented TGF-β1-induced GLα promoter activity, which
was inhibited by IFN-γ/Stat-1. Taken together, these results indicate that p300 acts as a basal element in GLα transcription
without binding to Smad3. Additionally, E1A, Stat1, and HDAC1 act as repressors of p300 in such transcription.
Keywordsp300-Germ-line α transcripts-IgA-TGF-β1-Smad3-FN-γ
[Show abstract][Hide abstract] ABSTRACT: IFN-gamma has been shown to either up- or down-regulate the expression of specific TGF-beta1-induced target genes. We investigated the effect of IFN-gamma on TGF-beta1-induced IgA isotype expression. We found that IFN-gamma inhibited not only TGF-beta1-induced germ-line (GL) alpha transcription, but also IgA secretion by TGF-beta1-stimulated murine B cells. Overexpression of Stat1 diminished TGF-beta1-induced, Smad3/4-and Runx3-mediated GL alpha promoter activity. Overexpression of p300 also increased the promoter activity, while its effect was abrogated by co-transfected Stat1. Stat1 interfered with the Smad3:p300 interaction, likely due to a stronger Stat1:p300 binding affinity. These results indicate that Stat1 can inhibit GL alpha transcription through binding to p300. Further, overexpression of SOCS1, a JAK inhibitor, diminished the antagonistic effect of IFN-gamma on TGF-beta1-induced GL alpha transcription and IgA secretion. These results indicate that JAK/Stat1-mediated IFN-gamma signaling antagonizes TGF-beta1-induced GL alpha transcription, mainly through deprivation of p300 from Smad3, resulting in decreased IgA synthesis.
[Show abstract][Hide abstract] ABSTRACT: APRIL (a proliferation-inducing ligand) is primarily expressed by macrophages and dendritic cells, and has profound effects on B cell physiology. In this study, we investigated the role of IL-4 in APRIL expression by mouse macrophages and the signaling mechanism involved. IL-4 markedly enhanced APRIL expression in mouse macrophages at the transcriptional and protein level. The p38MAPK inhibitor SB203580 completely abolished the IL-4 effect, whereas overexpression of CREB with IL-4 augmented APRIL expression. This increase was abolished by SB203580 treatment, indicating that p38MAPK may activate CREB. Overexpression of Stat6 also augmented IL-4-induced APRIL expression; this effect was partially abolished by SB203580 but not by the Jak inhibitor AG490, indicating that Stat6 mediates IL-4-induced APRIL expression in a Jak-independent manner and that p38MAPK acts as the intermediate. Our results demonstrate that IL-4 up-regulates APRIL expression through two divergent pathways in mouse macrophages, p38MAPK-CREB and p38MAPK-Stat6.
[Show abstract][Hide abstract] ABSTRACT: The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1, Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154, as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.
Full-text · Article · May 2009 · Nature Immunology
[Show abstract][Hide abstract] ABSTRACT: TGF-beta1 directs class switch recombination to IgG2b as well as IgA. We have shown that Smad3/4, Runx3, and p300 mediate TGF-beta1-induced germ-line (GL) gamma2b transcription and that there is a potential Smad-binding element (SBE, CAGAC, -38/-34) and Runx-binding element (TGTGGGT, +41/+47) in the promoter region. Here, we have characterized more putative transcription factor-binding elements in the promoter. Site-directed mutagenesis revealed that two more putative SBE (GTCTG, -67/-63 and +38/+42) are relevant to TGF-beta1-induced GLgamma2b promoter activity, a finding that was confirmed by EMSA. However, neither overexpression of Ets (i.e. Elf-1, Fli-1, or Pu.1) nor a mutation deleting a putative Ets-binding element (CAGGAA, -4/+2) affected basal or TGF-beta1-induced promoter activity. On the other hand, NF-kappaB repressed promoter activity without direct binding to two putative NF-kappaB-binding elements (GGACTCCCC, -63/-55; GGGCCTTTCC,+237/+246). Instead, NF-kappaB overexpression increased the expression of Smad7 transcripts. Moreover, p300 overexpression failed to rescue the inhibitory effect of NF-kappaB on GLgamma2b promoter activity. These results indicate that there are multiple SBE relevant to GLgamma2b promoter activity and that NF-kappaB acts in cooperation with p300 to downregulate promoter activity through increasing the gene expression of inhibitory Smad7.
Preview · Article · Apr 2009 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas(lpr/lpr) mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas(lpr/lpr) mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas(lpr/lpr) B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas(lpr/lpr) greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas(lpr/lpr) mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) theta, pol eta and pol zeta, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas(lpr/lpr) mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.
[Show abstract][Hide abstract] ABSTRACT: Activation-induced cytidine deaminase (AID) is an inducible gene that plays a critical role in Ig class switch recombination and somatic hypermutation in B cells. We explored the mechanisms by which IL-4 induces AID expression in mouse B cells. IL-4 increased AID expression and over-expression of Stat6 further augmented IL-4-induced promoter activity. The involvement of Stat6 in the promoter activity was confirmed using ChIP assays and site-directed mutagenesis. Treatment with H89, a PKA inhibitor, markedly decreased IL-4-induced AID expression, and over-expression of CREB enhanced it. These results indicate that Stat6 and PKA/CREB are involved in IL-4-induced AID expression. The relevance of these signal transducing molecules was verified using the TGFbeta1-induced IgA isotype switching model. Our results indicate that IL-4, through Stat6 and PKA/CREB, induces AID expression leading to Ig isotype switching event.
No preview · Article · Oct 2007 · Biochemical and Biophysical Research Communications