Abstract:The function of the pre-B cell receptor (pre-BCR) during B cell differentiation is not precisely defined. To investigate the pre-BCR receptor activity, we have established pre-BCR-positive pre-B cell lines that are able to differentiate into immature B cells in vitro. Antibody cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation. A specific tyrosine-phosphorylated 43 kDa protein (p43) was found down-stream of the pre-BCR. The results demonstrated the rece… Show more
“…We and others have found distinct calcium mobilization profiles upon aggregation of the pre-BCR, contrasting its downstream effects with those of the BCR (35,76,82,83). Unique proteins have also been detected following pre-BCR signaling, but have yet to be characterized (70). All of these data point to particular signaling functions of this receptor that contribute specifically to development.…”
The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (λ5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with λ5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.
“…We and others have found distinct calcium mobilization profiles upon aggregation of the pre-BCR, contrasting its downstream effects with those of the BCR (35,76,82,83). Unique proteins have also been detected following pre-BCR signaling, but have yet to be characterized (70). All of these data point to particular signaling functions of this receptor that contribute specifically to development.…”
The production of a mature B cell requires passage through a number of developmental checkpoints. The pre-BCR plays a critical role in passage through the pro-B cell/pre-B cell checkpoint, and thus plays a central role in regulating the differentiation of a B cell. Due to the significance of this receptor, it is imperative that pre-BCR expression and function are precisely regulated. In this study, we have investigated a system in which the regulation of the pre-BCR is altered. We have found that continued expression of components of the pre-BCR (λ5) resulted in a delay in the kinetics of B cell maturation. Pro-B cells from normal mouse bone marrow retrovirally infected with λ5 exhibited a delay in differentiation. As compared with wild-type cells at the same time point, there is a reduction in the presence of cell surface markers that indicate developmental progression, and there is a 6- to 16-fold decrease in the production of Ig-positive cells in B cell maturation assays. The capacity to alter B cell progression by modifying and extending pre-BCR expression argues that the receptor and its associated signals play a unique role in directing developmental outcomes.
“…Previously, we revealed that Ab cross-linking of the pre-BCR on the PreBR cell-line induced apoptosis and differentiation accompanied with tyrosine phosphorylation (35), while the immature B cell line, WEHI231, also undergoes apoptosis upon BCR crosslinking (40). Although PreBR cells express low and WEHI231 cells express high , the expression levels of Fc␥RIIB on both cell lines are equal.…”
Section: Discussionmentioning
confidence: 96%
“…PreBR and Fc␥ Ϫ/Ϫ PreBR cell lines were established from pro-B cells in the presence of IL-7 after removal of ST2 as described previously (35). These cell lines were cultured in SF-03 medium (Sanko Jyunyaku, Tokyo, Japan) containing 5 ϫ 10 Ϫ5 M 2-ME, 1ϫ nonessential amino acid solution (Life Technologies, Gaithersburg, MD), 0.03% primatone (Quest International, Naarden, The Netherlands), 2% FCS, and 100 U/ml rIL-7 (36) (a gift of Dr. Sudoh, Toray, Kamakura, Japan), as described previously (14).…”
Section: Animals and Cell Linesmentioning
confidence: 99%
“…We previously established pre-BCR ϩ pre-B cell lines (PreBR1 and PreBR2) (35). These cell lines had the large pre-BII phenotype, being positive for CD19, low , Ig, VpreB, 5, IL-7 receptor, and CD25, but negative for , CD23, c-kit, and CD40 ( Fig.…”
Section: Characterization Of the Pre-bcr ϩ Pre-b Cell Line Prebrmentioning
confidence: 99%
“…In these cell lines, Ab cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation (35). We show here that in a pre-BCR positive pre-B cell line from a Fc␥RIIB-deficient mouse, a high level of apoptosis was induced by both intact or F(abЈ) 2 anti-Ab cross-linking, and restoration of Fc␥RIIB to Fc␥RIIB-deficient cells blocked severe apoptosis following up-regulation of SHIP and Dok phosphorylation.…”
Many studies have shown that FcγRIIB is a negative regulator of B cell receptor signaling, and even though FcγRIIB is expressed through all developmental stages of the B cell lineage, its involvement in pre-B cell receptor (pre-BCR) signaling has not been examined. To investigate FcγRIIB function at the pre-B cell stage, we have established pre-BCR positive pre-B cell lines from normal mice and FcγRIIB-deficient mice, named PreBR and Fcγ−/−PreBR, respectively. These cell lines are able to differentiate into immature B cells in vitro by removal of IL-7. In PreBR, apoptosis was moderately induced by F(ab′)2 anti-μ Ab, but not by intact anti-μ Ab. Phosphorylation of SH2-containing inositol 5-phosphatase (SHIP) and Dok, which are involved in FcγRIIB signaling, was induced by anti-μ cross-linking in PreBR. In contrast, apoptosis was strongly induced by both the F(ab′)2 and intact anti-μ Abs in Fcγ−/−PreBR, and the level of phosphorylation of SHIP or Dok was much lower in Fcγ−/−PreBR than those observed in PreBR. Restoration of FcγRIIB to Fcγ−/−PreBR followed by anti-μ cross-linking blocked severe apoptosis, and up-regulated SHIP and Dok phosphorylation. The results demonstrate that FcγRIIB negatively regulates pre-BCR-mediated signaling for apoptosis.
Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding site for early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBFregulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.
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