Efficiency of Iε promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightlylinked to the strength of transcription

Zhang, Ke; Zhang, Ling; Yamada, Tetsuji; Lee, Anna; Saxon, Andrew

doi:10.1002/1521-4141(200202)32:2<424::aid-immu424>3.0.co;2-p

Cited by 7 publications

(14 citation statements)

References 23 publications

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“…RNA suspended in 0.1% diethylpyrocarbonate-treated water was digested with DNase I (Sigma) to remove possible contaminating DNA and was then extracted with phenol/chloroform followed by precipitation in ethanol. Total RNA (1 g) was reverse-transcribed to cDNA as described previously (17). All PCR assays were done in 50-l reaction volumes containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 mM MgCl 2 , 1 M of each primer, and 2.5 units of Taq polymerase (Promega).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fcγ-Fcϵ Chimeric Protein

Yamada

Zhu²,

Zhang³

et al. 2003

Journal of Biological Chemistry

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Immunoglobulin E (IgE) is important in mediating human allergic diseases. We tested the hypothesis that a human Ig Fc␥-Fc⑀ bifunctional chimeric protein, GE2, would inhibit IgE class switch recombination (CSR) by co-aggregating B-cell CD32 and CD23. Indeed, GE2 directly inhibited ⑀ germ-line transcription, subsequent CSR to ⑀ and IgE protein production. This CSR inhibition was dependent on CD23 binding and the phosphorylation of extracellular signal-related kinase (ERK), and it was mediated via suppression of interleukin-4-induced STAT6 phosphorylation. Treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase 1 (MAPKK1 (MEK1)) and MEK2 reversed the ability of GE2 to decrease CSR and STAT6 phosphorylation. GE2 stimulation induced ERK phosphorylation, whereas it did not alter the phosphorylation of c-Jun N-terminal kinase or p38 MAPK. The ability of GE2 to block human isotype switching to ⑀, in addition to its already demonstrated ability to inhibit mast cell and basophil function, suggests that it will provide an important novel benefit in the treatment of IgE-mediated diseases.

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Section: Methodsmentioning

confidence: 99%

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fcγ-Fcϵ Chimeric Protein

Yamada

Zhu²,

Zhang³

et al. 2003

Journal of Biological Chemistry

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“…Human B cell lines BL-2, DG75, and Ramos 2G6 were maintained and cultured with the stimulation indicated in each figure. For transient transfection, 1 ϫ 10 6 DG75 cells in 0.2 ml of medium were electroporated (200V, 975 F) with 20 g pST6-DN or with control plasmids as described previously (37).…”

Section: Cells Cell Lines and Transfectionmentioning

confidence: 99%

“…Total RNA was extracted by TRIzol reagent (Invitrogen, San Diego, CA) as described (37). RNA was digested with DNase I (Sigma-Aldrich, St. Louis, MO) to remove contaminating DNA.…”

Section: Rt-pcrmentioning

confidence: 99%

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Human Activation-Induced Cytidine Deaminase Is Induced by IL-4 and Negatively Regulated by CD45: Implication of CD45 as a Janus Kinase Phosphatase in Antibody Diversification

Zhou

Saxon

Zhang

2003

The Journal of Immunology

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Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and VH gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.

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“…Switch recombination was quantified employing a recently established switch vector assay (20), S-S⑀ recombination in primary B cells was measured by digestion-circularization-PCR (DC-PCR), and ⑀GTs were assessed by RT-PCR. We also examined the effect of ␣CD45 on IL-4-and ␣CD40-driven phosphorylation and activation of JAK1, JAK3, and STAT6, as well as phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular-signal related kinase (ERK).…”

mentioning

confidence: 99%

CD45 Controls Interleukin-4-mediated IgE Class Switch Recombination in Human B Cells through Its Function as a Janus Kinase Phosphatase

Yamada¹,

Zhu²,

Saxon³

et al. 2002

Journal of Biological Chemistry

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CD45 plays a critical regulatory role in receptor signaling through its protein tyrosine phosphatase and Janus kinase (JAK) phosphatase activities. To investigate whether CD45 also plays a regulatory role in Ig class switching in human B cells, we examined the effects of CD45 triggering on Ig class switching to IgE and its relationship with CD45 JAK phosphatase activity. Anti-CD45 triggering of CD45 significantly inhibited interleukin-4 ؉ anti-CD40-induced switch recombination in a switch recombination vector assay in stably transfected Ramos 2G6 human B cells, as well as Ig ⑀ germ-line transcription and S-S⑀ switch recombination in primary human B cells. These negative regulatory effects on Ig class switching were concomitant with the ability of CD45 to dephosphorylate the induced phosphorylation of JAK1, JAK3, and signal transducer and activator of transcription 6, but not on stress-activated/mitogenactivated protein kinases. We also showed that phosphorylated JAK1 and JAK3 were directly dephosphorylated by recombinant CD45 in vitro. These results indicate that CD45 is able to function as JAK phosphatase in human B cells and that this activity is directly associated with the negative regulation of the class switch recombination to IgE. CD45 may be an appropriate target drug for modulating IgE in allergic diseases.

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Efficiency of Iε promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightlylinked to the strength of transcription

Cited by 7 publications

References 23 publications

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fcγ-Fcϵ Chimeric Protein

Inhibition of Interleukin-4-induced Class Switch Recombination by a Human Immunoglobulin Fcγ-Fcϵ Chimeric Protein

Human Activation-Induced Cytidine Deaminase Is Induced by IL-4 and Negatively Regulated by CD45: Implication of CD45 as a Janus Kinase Phosphatase in Antibody Diversification

CD45 Controls Interleukin-4-mediated IgE Class Switch Recombination in Human B Cells through Its Function as a Janus Kinase Phosphatase

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