The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.-C.
Right arrow Articles by Muthusamy, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.-C.
Right arrow Articles by Muthusamy, N.
The Journal of Immunology, 2006, 176: 2208-2218.
Copyright © 2006 by The American Association of Immunologists

Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival1

Hui-Chen Chen{dagger}, John C. Byrd*,§,¶ and Natarajan Muthusamy2,*,{dagger},{ddagger},¶

* Division of Hematology and Oncology, Department of Internal Medicine, {dagger} Molecular Virology, Immunology and Medical Genetics, {ddagger} Veterinary BioSciences, § Division of Medicinal Chemistry, College of Pharmacy, and OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser119-ala phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed ~70% increase in pre-B1 (CD43+B220+CD24+(int)) and ~60% decreased pre-BII (CD43+B220+CD24++(high)) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed ~4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2–/– mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43+B220+CD24high population that is normally absent in RAG-2–/– mice. In the periphery, the Tg mice exhibited decreased CD21dimCD23highIgM+ follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cyclic AMP response element binding protein-1 belongs to the basic leucine zipper family of transcription factors. CREB-1 binds to CRE, which is composed of TGANNTCA sequences as homo- and heterodimers in association with members of the CREB/activating transcription factor (ATF)3 family of proteins, including ATF-1 and cAMP response element modulator, as well as AP-1 family members such as c-Jun (1, 2, 3, 4, 5). The activation-induced phosphorylation of CREB-1 at Ser133 (or Ser119) in an alternatively spliced form increased the DNA-binding activity of the protein in some but not other cell types (4, 5, 6, 7). Phosphorylation at Ser133 activates CREB-1, at least in part, by facilitating its binding to CREB-binding protein (CBP). This, in turn, interacts with and activates the components of basal transcriptional machinery (8, 9, 10, 11, 12, 13). Multiple signaling pathways, including cAMP-responsive protein kinase A, protein kinase C, calcium/calmodulin-dependent CaM kinases II and IV, and a Ras-dependent serine/threonine kinase RSK2, mediate phosphorylation and activation of CREB-1 in different cell types (14, 15, 16, 17, 18, 19, 20, 21, 22).

Activation of resting murine B and T cells through antigen receptors leads to the phosphorylation of CREB-1 at critical Ser133, which is essential for the transcriptional activation function of the protein (22, 23, 24). Stimulation through the BCR resulted in a dose-dependent induction of CREB-1-binding activity that is subjected to negative regulation by IFN-{gamma} in a STAT-1-dependent manner (25). Consistent with a role for CREB-1 in the regulation of cell growth, the expression of immediate early growth response genes, such as c-fos and junB, and cell cycle regulatory genes, such as the proliferating cell nuclear Ag (PCNA), is regulated by Ser133-phosphorylated CREB-1 through direct binding and activation of the promoter region of these genes (26, 27, 28, 29, 30).

A requirement for CREB-1 in mature B cell survival is implicated by the CREB-1-dependent induction of the bcl-2 gene in human B cell lines and transgenic (Tg) mice (31, 32, 33). Ser133-phosphorylated CREB-1 binds to the CRE element in promoter regions of antiapoptotic genes such as Bcl-2 and Mcl-1 and regulate their expression. CREB-1–/– mice die shortly after birth, thus precluding the analysis of the immune function of mature B cells (34). The lack of a strong phenotype in some CREB family member knockout studies suggested the functional redundancy among the members of this family (35). To determine the in vivo role of CRE-binding proteins in early B cell development and functional maturation, we generated four independent Tg mice lines overexpressing a dominant-negative phosphomutant CREB-1 in developing B cells in the bone marrow. Characterization of these Tg mice revealed a critical role for CRE binding proteins in different stages of B cell development and functional maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Mutant CREB-1-Tg animals described were generated at the OSU CCC/CCRI transgenic facility. The Tg expression vector pBH was constructed by cloning the EcoRI fragment of pEPB splice-neo (a gift from Drs. W. Muller and K. Rajewsky, Institute for Genetics, University of Cologne, Cologne, Germany) containing the Ig H chain promoter and intronic enhancer and the SV40 splice and poly(A) into the EcoRI site of pBluescript KS II(+). The dominant-negative CREB-1 cDNA was produced by PCR-mediated mutagenesis of the human {Delta}CREB-1 cDNA at Ser119 (Ser119 to Ala) (30). The hCREB-1Ser119-Ala was cloned into the BglII site of the pBH Tg vector. Four independent founder lines were identified by PCR using primers 5'-AAAACCACTTCTTCAAACCACAGC-3' and 5'-CTGCTGGAGAAGAAGGGACATC-3' corresponding to the Ig H chain intronic enhancer and the Ig H chain promoter, which amplified ~700 bp fragment in the Tg tail DNA. The lines were further confirmed by Southern blot analysis. Recombination activating gene-2-deficient (Rag-2–/–) mice were obtained originally from Dr. F. Alt (Harvard Medical School, Boston, MA). Female FVB/N mice were obtained from Harlan Sprague Dawley. All animal studies described were approved by the Institutional Animal Care and Use Committee.

Reagents

Goat anti-mouse IgM (Jackson Immuno Research Laboratories) and PMA, ionomycin, and DNP conjugated to keyhole limpet hemocyanin (KLH) were purchased from Calbiochem. [32P]dCTP and dGTP were obtained from Amersham Life Sciences. Rabbit complement was purchased from Pel-Freeze Biologicals. Ficoll-Paque, LPS, trinitrophenyl (TNP)-LPS, and Hoechst no. 33342 were purchased from Sigma-Aldrich. Anti-human CREB-1 Ab (24H4B) was obtained from Santa Cruz Biotechnology. The LumiGLO chemiluminescent kit was purchased from Kirkegaard & Perry Laboratories. Fluorochrome-labeled anti-B220 (RA3-6B2), anti-IgM (R6-60.2), anti-MAC-1 (M1/70), anti-IgD (AMS9.1), anti-CD5 (53-7.3), anti-CD43 (S7), anti-CD24 (30-F1), anti-CD4 (RM4–5)m and anti-CD8 (53-6.7) were purchased from BD Pharmingen. The Vybrant CFDA SE cell Tracer kit (V12883) was purchased from Molecular Probe.

Southern blot analysis

The tail biopsies from Tg or nontransgenic (NTg) mice were digested overnight with lysis buffer (50 mM Tris (pH 8), 100 mM EDTA, 0.5% SDS, and 350 µg of proteinase K), and the genomic DNA samples were prepared by phenol/chloroform extraction followed by ethanol precipitation. Genomic DNA (5–10 µg) digested with EcoRI was separated on 0.7% agarose gel, transferred onto Immunobilon-Ny+ transfer membrane (Millipore), cross-linked by UV, and then baked in a vacuum oven at 80°C for 30 min. The membrane was subsequently prehybridized for 30 min at 42°C in hybridization solution (2.5 mg of salmon sperm DNA, 50% formamide, 4.8x SSC, 8 mM Tris (pH 7.5), 5x Denhardt’s solution, 0.2% SDS, and 5% dextran sulfate), followed by fresh hybridization solution containing denatured radiolabeled probe (a transgene-specific 0.35-kb PstI/BamHI fragment spanning the SV40 splice and poly(A) region of the Tg construct) overnight. The blots were then washed and exposed to x-ray film.

Northern blot analysis

RNA was isolated using TRIzol reagent. Total RNA (10 µg) was separated on 1% RNA agarose gel, transferred onto nylon Hybond-N+ membrane (Amersham Biosciences), and then cross-linked by UV. The membrane was then baked in a vacuum oven at 80°C for 30 min and prehybridized for 2 h at 42°C in hybridization solution (5% standard saline citrate phosphate/EDTA, 5% Denhardt’s solution, 0.5% SDS, 50% formamide, 10% dextran sulfate, and 3 µg of salmon sperm DNA), followed by fresh hybridization solution containing a denatured radiolabeled transgene-specific 0.3-kb XhoI/BamHI fragment derived from the CD2 poly(A) region.

Preparation of nuclear extract

Splenic B cells from Tg and NTg mice were washed in cold PBS, resuspended in 100 µl of buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF/aprotinin), and incubated on ice for 10 min and centrifuged. The cell pellet was resuspended in 25 µl of buffer C (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF/aprotinin) and incubated on ice for 20 min and centrifuged at 12,000 rpm for 2 min at 4°C. The concentration of the nuclear protein in the supernatant was determined by the Bradford assay (Bio-Rad). The nuclear extract was aliquoted into (5 µl) volumes and frozen at –70°C until further use.

Double-stranded oligo probe preparation and EMSA

The following double-stranded oligonucleotides were used: CRE oligonucleotide (CRE element underlined), 5'-gatcGCCTCCTTGGCTGACGTCAGAGAG-3' (forward); and mutant CRE oligonucleotide (mCRE), 5'-gatcGCCTCCTTGGCTCAGCACAGAGAG-3' (forward). Annealed double-stranded oligo nucleotides (100 ng) were labeled with 32p-nucleotides using nick translation buffer as described previously (30). The free probe was removed by purification in G50 Sephadex spin columns. The binding reactions were conducted, at room temperature, with 3 µg of nuclear extract, 30,000 dpm (0.1–0.5 ng) of radiolabeled oligonucleotide probe, in 5x Ficoll-binding buffer (10 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and 4% Ficoll), 250 ng of poly(deoxyinosinic-deoxycytidylic acid) in 75 mM KCl and double-distilled H2O to make the volume to 15 µl. The DNA-protein complexes were fractionated by electrophoresis in 4% nondenaturing polyacrylamide gel, run in 0.25x Tris-borate-EDTA, at 4°C. The gel was then dried on 3M Whatman paper and subjected to autoradiography. In vitro transcribed and translated CREB-1 protein generated using rabbit reticulolysate system was used as a positive control (30).

Cell preparation and culture

Mice (4–8 wk old) were sacrificed by cervical dislocation. Bone marrow cells were obtained from the femur. Peritoneal cells were collected by flushing the peritoneal cavity with PBS. Splenic B cells were prepared by incubating the RBC-depleted splenocytes for 20 min on ice in an anti-Lyt2 (3.155) and anti-L3T4 (R7-172.4) Ab mixture, followed by 30-min incubation with rabbit complement at 37°C. The cells were then washed and resuspended at 107 cells/ml in RPMI 1640 medium. The live B cells were purified using Ficoll-Paque density gradient method. Single-cell suspensions were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated FCS, 50 µM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mM nonessential amino acid. The cells were cultured in a 37°C incubator with 5% CO2.

Western blot analysis

The total cellular protein was extracted by boiling 4 x 106 cells in 50 µl of 1x SDS sample buffer. The samples were separated on 10% SDS-polyacrylamide gel by electrophoresis and blotted onto a nitrocellulose membrane (Schleicher & Schuell Microscience). After blocking with PBS containing 5% nonfat dry milk powder, the blots were probed with 1 µg/ml mouse-anti-human CREB-1 Ab. After washing the membrane three times with PBS + 0.1% Tween 20, the blots were further incubated with the appropriate goat anti-mouse Ig-HRP-conjugated secondary Ab. The blots were developed using LumiGLO Chemiluminescent Substrate System as per the manufacturer’s instructions.

Flow cytometry

Single-cell suspensions from the bone marrow, spleen, lymph node, thymus, or peritoneum were washed twice with PBS containing 0.1% BSA. Cells were incubated with indicated fluorochrome-labeled Abs on ice for 30 min, washed twice, and then resuspended in PBS containing 0.02% sodium azide. Dual and multiparameter flow cytometric analysis was performed on ELICS ELITE ESP Coulter Counter. The data were acquired in list mode and analyzed using the Windows Multiple Document Interphase (WinMDI) program developed by J. Trattor at The Scripps Institute (La Jolla, CA). The pre-BI and pre-BII B cells were sorted using FACS following staining with indicated fluorochrome-labeled Abs.

RT-PCR analysis

RNA samples from sorted pre-BI or pre-BII cells were isolated using TRIzol reagent with 20 µg of glycogen as the carrier. RNA (100–200 ng) was used for first-strand cDNA synthesis. The reverse transcription was performed using random primer oligonucleotides as primers and mouse mammary tumor virus reverse transcriptase as described in the manufacturer’s protocol (Invitrogen Life Technologies). One microliter of reverse transcriptase product (cDNA) was amplified using primer pairs specific for tested genes. PCR were performed in a 25-µl volume containing 1 µl of cDNA, 1x PCR buffer, 200 µg of four deoxynucleotide triphosphates, 6 µM of each of the sense and antisense primers, 2 mM MgCl2, and 2.5 U of Taq polymerase (Invitrogen Life Technologies), using PTC-100 Programmable Thermal Controller (MJ Research). The PCR cycle was repeated 26–32 cycles. The PCR products were separated on 1.5% agarose gel containing the ethidium bromide and photographed, or a semiquantitative multiplex RT-PCR in the same tube was designed to compare the RT-PCR products of genes of interest along with hypoxanthine phosphoribosyltransferase (HPRT) gene products to determine the relative levels of expression of these genes in each of these samples. The density of each band was quantified using ImageQuant 5.0 (Molecular Dynamics). All the oligonucleotide primers were designed according the published sequences of the respective genes. The primers were designed to be specific to the respective genes and do not possess a significant match to any other mouse sequence in the GenBank database. The oligonucleotide primers were custom-synthesized by Invitrogen Life Technologies. The primer sequences used for PCR, with the expected product size in brackets are listed as follows: HPRT (176 bp) sense, 5'-CCAGCAAGCTTGCAACCTTAACCA-3', and antisense, 5'-GTAATGATCAGTCAACGGGGGAC-3'; Mb-1 (310 bp) sense, 5'-GCCAGGGGGTCTAGAAGC-3', and antisense, 5'-TCACTTGGCACCCAGTACAA-3'; c-Jun (692 bp) sense, 5'-GGGGAAGCACTGCCGTATGGAG-3', and antisense, 5'-CCCGGGTTGAAGTTGCTGAGGTT-3'; c-Myc (506 bp) sense, 5'-CTCGCCGCCGCTGGGAAACTT-3', and antisense, 5'-CACCGCCGCCGTCATCGTCTT-3'; JunB (660 bp) sense, 5'-CGCCCGGATGTGCACGAAAATG-3', and antisense, 5'-CGGAAGCGCCACGACTCAAACC-3'; c-Fos (584 bp) sense, 5'-GGGTTTCAACGCCGACTACGA-3', and antisense, 5'-GGGCTGCCAAAATAAACTCCA-3'; PCNA (800 bp) sense, 5'-TCCTTGGTACAGCTTACT-3', and antisense, 5'-TGCTAAGGTGTCAGCATT-3'; and VpreB (376 bp) sense, 5'-CTGCTGTCCTGCTCATGCT-3', and antisense, 5'-ACGGCACAGTAATACACAGCC-3'.

Cell cycle analysis

Bone marrow cells (4 x 106) were stained with anti-B220-R-PE-cyanine 5 (PE-Cy5, BD CyChrome), anti-CD43-R-PE, and anti-CD24-FITC for 20 min on ice and subsequently washed twice with PBS-0.01% BSA buffer. The cells were fixed in 1% paraformaldehyde for 10 min, washed twice with PBS, and then permeabilized with freeze-cold 70% ethanol for 10 min on ice. The cells were spun down and resuspended in 2 ml of 2 µg/ml Hoechst 33342 staining solution for 30 min at room temperature. The cell cycle analysis was performed on ELICS ELITE ESP flow cytometer. The data were acquired in list mode and analyzed using WinMDI version 2.8 and Modfit LT cell cycle analysis software.

B Cell proliferation assay

Purified splenic B cells (2.5 x 105) were cultured in 96-well tissue culture plates (Costar), with 10 µg/ml goat anti-mouse IgM and 0.1 µg/ml PMA ± 0.5 µg/ml ionomycin as indicated. Cells were cultured for 40 h in 5% CO2 at 37°C and then pulsed with 1 µCi of [3H]thymidine (sp. act. 2Ci/mM) (Amersham Biosciences) for 8 h. [3H]Thymidine incorporation was analyzed in a Packard liquid scintillation counter (Packard). Representative results of multiple experiments are presented as geometric mean of responses from triplicate cultures with SE of mean.

Immunization and ELISA

Groups of four to six wild-type and mCREB-1 Tg mice (6–8 wk old) were immunized i.p. with 50 µg of DNP-KLH, 50 µg of TNP-LPS, or 10 µg of TNP-Ficoll in sterile PBS. The mice were bled before (preimmune sera) and after immunization at weekly intervals. Four weeks after the primary immunization with DNP-KLH, the mice were challenged with 50 µg of DNP-KLH and were bled 5 days later for secondary immune sera. Anti-TNP Abs were determined using isotype-specific ELISA as described previously (36). Each of the wells in the ELISA plates were coated with 20 µg of TNP and blocked with 10% FBS in PBS. Sera were serially diluted, and the Ag specific Ab titer was detected with peroxidase-conjugated rabbit anti-mouse isotype-specific Abs using SBA clonotyping system (Southern Biotechnology Associates). The results are presented as mean serum concentrations of indicated isotypes ± SD from 5 to 10 mice/group.

Adoptive transfer analysis

Groups of four recipient RAG-2–/– mice were sublethally irradiated with 300 rad from a 137Cs source. Donor bone marrow cells (10 x 106) suspended in PBS were injected i.v. into the recipient animals 1 day after irradiation. Four weeks after the transfer, the bone marrow and splenic cells were analyzed by flow cytometry. The donor-derived cells in the recipients were identified by CD43+B220+CD24high populations that are not normally found in RAG-2–/– mice (37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Production of Tg mice overexpressing dominant-negative mutant CREB-1 (CREB-1Ser119->Ala) in bone marrow

Activation of B cells through the AgR (BCR) induced phosphorylation of CREB-1 on the Ser119 residue that is known to be required for the transcriptional activation of CREB-1 (15). We have shown recently that activation of B cells through BCR induced dose-dependent CRE-binding activity, with minimal alteration in the levels of the CREB-1 protein. Furthermore, BCR induced CREB-1-binding activity is subjected to IFN-{gamma}-mediated regulation in B cells. These observations along with the constitutive expression pattern of CREB-1 in the bone marrow B cells suggested a potential role for CREB-1 in B cell development and/or functional maturation (Ref.25 ; H.-C. Chen and N. Muthusamy, unpublished observation). To test this directly, we generated Tg mice overexpressing a dominant-negative CREB-1 transcription factor in developing B cells in the bone marrow. Schematic representation of a Tg vector-encoding the mutant CREB-1 in which the critical Ser119 had been changed to alanine is shown in Fig. 1a. Previous studies have shown that such a mutant CREB-1 protein bound to DNA but abrogated transcription activation of target genes, thus serving as a dominant-negative inhibitor of CREB-1 in vitro and in vivo (8, 28, 30, 32). Human {Delta}CREB-1 cDNA with Ser119 to alanine mutation was generated by PCR and cloned into a B cell-specific pBH expression vector containing the Ig µ H chain promoter and intronic enhancer (Fig. 1a). Southern blot analysis of tail DNA from four independent founders, using a probe from the unique region of the Tg vector shown in Fig. 1a, identified ~5, 13, 38, and 15 copies of the transgene (Fig. 1b). A representative Northern blot analysis of total RNA isolated from the bone marrow of each of the Tg lines and a NTg littermate from one of the lines using the human CREB-1-specific probe is shown in Fig. 1c. The expression of the human CREB-1 transgene was confirmed by Western blot analysis of the protein isolated from each of the Tg lines using anti-human CREB-1 Ab (Fig. 1d) and by EMSA (Fig. 1e).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Production of Tg mice with dominant-negative mutant CREB-1 transgene. a, Schematic representation of the CREB-1A119 transgene. The Ig H chain promoter, enhancer, and the SV40 splice and poly(A) site are shown. The mutant hCREB-1 was produced by PCR-mediated mutagenesis of the human {Delta}CREB cDNA at Ser119 (Ser119 to Ala119), thereby creating a nonphosphorylatable dominant-negative form of CREB-1 (CREB-1A119). The hCREB-1A119 was cloned into the BglII site of the pBH transgene vector as described in Material and Methods. b, Southern blot analysis of tail DNA from wild-type (WT) and four independent Tg founder lines. Tail DNA was probed with probe unique to the Tg vector (shown in a). The numbers shown in the bottom of each lane represent the different founder lines and WT. c, Northern blot analysis of RNA from bone marrow cells from WT and four different founder lines of Tg mice. Expression levels of the transgene transcript were identified using RNA prepared from bone marrow cells probed with probe specific to the transgene as described in Materials and Methods. Top panel, The expression of transgene transcripts. A comparable 28s ribosomal RNA level in each of the lanes is shown as loading control (bottom panel). The numbers shown in the bottom of each lane represent the different founder lines and WT control. d, Western blot analysis of transgene expression in bone marrow cells from WT and four different founder lines of Tg mice. The expression level of Tg human CREB-1 protein was analyzed in protein extract prepared from WT and four different founder lines of Tg mice, using an Ab specific to human CREB-1. The presence of equivalent levels of protein loading in each of the lanes is shown (control). The numbers shown in the bottom of each lane represent the different founder lines and WT control. e, EMSA of nuclear extract from NTg and Tg mice. Nuclear extract prepared from splenic B cells from two Tg (Tg#1 and Tg#2) and a NTg mice were analyzed by EMSA using radiolabeled WT CRE (lanes 2–4) or mutant CRE (lanes 5–7) oligonucleotides as described in Materials and Methods. In vitro transcribed and translated recombinant CREB-1 protein (IVT-CREB) was used as positive control (lane 1).

 
Defective development of mature B cells associated with pre-BI to pre-BII transitional block in bone marrow of mCREB-1 Tg mice

Expression of CREB-1 throughout the B cell developmental stages suggested a role for CREB-1 in B cell development. To directly test whether overexpression of dominant-negative CREB-1 in the bone marrow interfered with normal B cell development, bone marrow cells from Tg and NTg littermates were analyzed by flow cytometry using fluorochrome-labeled anti-IgM and anti-B220 Abs. The B220+/IgM– population, which represents the pro-B and pre-B population, was decreased ~65% in the Tg bone marrow. (7.56 ± 0.89% in NTg mice vs 3.5 ± 1.1% in Tg mice; Fig. 2a and Table I). Furthermore, in comparison to the NTg littermates, the mCREB-1 Tg mice revealed ~65% decrease in B220+/IgM+ B cells that represent the immature and mature B cells in the bone marrow (4.4 ± 0.98% in NTg vs 1.4 ± 0.19% in Tg mice (Fig. 2a, left panel, and Table I)). This defective development is unique to B cells as no detectable defects in the development of T cells in the thymus, spleen, or lymph node in Tg mice were observed (Fig. 2a, right panel, and data not shown). Development of B cells from lymphoid precursors in the bone marrow can be distinguished into pro-B (B220+CD43+CD24–), pre-BI (B220+CD43+CD24+(int)), early pre-BII (B220+CD43+CD24++(high)), late pre-BII (B220+CD43–CD24++(high)), immature (B220+CD43–CD24++IgM+), and mature (B220+CD43–CD24–IgM+) B cell stages based on the differential expression of B220, CD43, and CD24 surface molecules. Decreased B220+IgM+ and B220+IgM– B cell in the bone marrow suggested a possible developmental block at or before the sIgM expressing immature B cell stage. To define the specific stage at which overexpression of mCREB-1 results in early B cell developmental defects, bone marrow cells from Tg and NTg littermates were examined by multiparameter flow cytometry using anti-B220 (CyChrome), anti-CD43 (PE), and anti-CD24 (FITC) Abs. As shown in Fig. 2b, ~70% decrease in B220+CD43– cells and a moderate, yet consistent decrease (~15%) in B220+CD43+ cells was observed. Thus, B cell development appears to be blocked as early as the B220+CD43+ stage in the Tg mice. The B220+CD43+ cells can be further distinguished into pro-B (B220+CD43+CD24–), pre-BI (B220+ CD43+CD24+(int)), and early pre-BII (B220+CD43+CD24++(high)) B cells based on the expression of CD24 (38, 39). Although three-color flow cytometric analysis revealed no difference in B220+CD43+CD24– cells, ~70% increase pre-BI cells (39% in NTg vs 67% in Tg mice), and ~60% decrease in pre-BII cells (46% in NTg vs 20% in Tg mice) was observed in the bone marrow from Tg mice (Fig. 2, b and c, and Table I), indicating a developmental block in pre-BI to pre-BII transition in mCREB-1 Tg mice.


Figure 2
View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 2. Defective pre-BI to Pre-BII developmental progression in mCREB-1 Tg mice. a, left panel, Bone marrow cells from NTg and Tg littermates were stained with PE-conjugated anti-B220 and FITC-conjugated anti-IgM. Right panel, Thymocytes from NTg and Tg littermates were stained with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8. The results shown are representative of more than eight experiments. The cells representing various populations are presented as percentage in each of the quadrants. b, Bone marrow cells from NTg (top panel) and Tg (lower panel) littermates were stained with CyChrome-conjugated anti-B220, PE-conjugated anti-CD43, and FITC-conjugated anti-CD24 Abs. The expression levels of CD24 in the B220+CD43+ cells are shown in the right of the panel. Pro-B cells (B220+CD43+CD24–), pre-BI cells (B220+CD43+CD24+(int)), and early pre-BI cells (B220+CD43+CD24++) are distinguished based on the expression levels of CD24 in the B220+CD43+ cells. The numbers represent the percentage of cells in bone marrow (left panel) and the percentage of cells in the B220+CD43+ cells (right panel). The results shown are representative of four independent experiments. c, The percentage of pre-BI and early pre-BII in the bone marrow from NTg and Tg littermates were analyzed as mentioned in b. The numbers represent the mean of percentage of cells ± SD from four mice. (*, p < 0.01; **, p < 0.02; unpaired Student’s t test). d, Decreased bone marrow cells in the Tg mice. The total bone marrow cells from NTg and Tg mice femur were enumerated using a hemocytometer from four mice. Numbers represent mean ± SD. Right panel, X-ray analysis (top) and histological analysis (H&E staining) of Tg and NTg littermates exhibiting dense skeletal structures in the Tg mice compared with NTg littermates. e, Bone marrow cells (10 x 106/mice) from NTg and Tg mice were injected i.v. into sublethally irradiated (300 rad) RAG-2–/– mice. Four weeks after the transfer, B cells in the bone marrow from the recipients were analyzed by FACS as mentioned in b. The fold change in the percentage of B220+CD43+CD24high (early pre-BII cells) in the recipients compared with the control mice are shown. {square}, The control RAG-2–/– mice without transfer with the cells (PBS control). {blacksquare}, The recipient mice with NTg bone marrow cells. {cjs2108}, The recipient mice with Tg bone marrow cells.

 

View this table:
[in this window]
[in a new window]
  		Table I. B cell populations in bone marrow of mCREB Tg micea

 
The developmental defect in mCREB-1 bone marrow is cell intrinsic

In addition to the B cell developmental defects, significant reduction in the bone marrow cells due to development of osteopetrosis was observed in Tg mice (29.6 x 106± 4.6 x 106 in NTg vs 5.9 x 106± 1.6 x 106 in Tg mice) (Fig. 2d) (M. Bayoumy, H.-C. Chen, and N. Muthusamy, manuscript in preparation). The bone marrow environment is critical for B cell development. To determine whether the developmental block observed in the mCREB-1 Tg bone marrow B cells is due to cell intrinsic defects, bone marrow cells from either wild-type or Tg littermates were adoptively transferred i.v. into sublethally irradiated RAG-2–/– mice. RAG-2–/– mice lack B220+CD43+CD24high population due to developmental block at pro-B/pre-BI stage of the B cell development (37). Analysis of B220+CD43+CD24high pre-BII population in the RAG-2–/– recipients 4 wk after transfer exhibited ~2-fold increase in B220+CD43+CD43high pre-BII cells in mice transferred with the wild-type bone marrow cells. However, no increases in B220+CD43+CD24high cells were observed in the Tg bone marrow recipients (Fig. 2e). Furthermore, consistent with the defective B cell progression, RAG-2–/– recipients that received the Tg bone marrow cells exhibited ~50% reduction in more mature B220+CD43– B cells compared with those that received the same number of wild-type bone marrow cells (data not shown). No osteopetrosis was observed in the recipient RAG-2–/– mice in the 4 wk time period of the study.

Deregulated expression of c-Jun and JunB associated with defective S-phase entry of pre-BII B cells in the mCREB-1 Tg mice

PhosphoCREB-1 has been shown to be involved in regulation of bcl-2 gene expression in human B cell line and murine B cells (32, 33). RT- PCR analysis of RNA from Tg and NTg pre-BI and Pre-BII cells revealed comparable levels of bcl-2 transcription. It is likely that the B cell developmental abnormalities in the mCREB-1 Tg mice could be due to potential abnormal down-modulation of bcl-2 in other stages of developing B cells. To test this directly, mCREB-1 mice were crossed with Tg mice that overexpress bcl-2 in B cells. Overexpressed Bcl-2 failed to rescue the bone marrow B cell developmental abnormalities in the double Tg mice (data not shown), suggesting bcl-2-independent CREB-1-dependent regulatory pathways in the observed B cell developmental defect. Expression of immediate early cell growth regulatory genes such as c-fos, junB, and PCNA have been shown to be regulated by Ser119/133-phosphorylated CREB-1 (27, 28). Furthermore, protein kinase-mediated signaling events have also been shown to regulate c-Jun, JunB, and JunD (40, 41, 42). To test whether deregulation of any of these potential phospho-CREB-1 target genes could contribute for the defective expansion of pre-BII cells in the Tg mice, RNA from sorted pre-BII B cells from the bone marrow of Tg and NTg littermates were analyzed by reverse transcriptase-mediated PCR analysis. Interestingly, pre-BII B cells from Tg mice revealed a consistent increase in c-Jun and JunB transcripts compared with wild-type littermate controls (Fig. 3a). No detectable differences were observed in expression of PCNA, Mb-1, and vpreB or HPRT transcripts (Fig. 3a). The increased junB and c-jun expression was confirmed independently by multiplex PCR analysis using same RNA preparations analyzed with respective test gene primers along with HPRT internal control primers. A representative result of this analysis and summary of four independent experiments are shown in Fig. 3b.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. Increased junB and c-jun expression associated with defective S phase entry of Tg pre-BII B cells. a, Total RNA was prepared form sorted pre-BII cells using the TRIzol method. The expression levels of indicated gene transcripts were determined by RT-PCR analysis as mentioned in the Materials and Methods. HPRT was used as an internal control to normalize the expression levels of the genes tested. The data shown here are representative of four independent experiments. b, Relative levels of junB and c-jun expression in NTg and Tg pre-BII B cells. Multiplex RT-PCR analysis of JunB and c-Jun transcripts using the respective primers and HPRT primer sets in the same reaction to eliminate variabilities is shown on the top two panels. The mean fold changes relative to the NTg controls (set at 1) in four independent experiments is shown in the bottom panels. (*, p < 0.001, and **, p < 0.01, when compared with NTg controls). HPRT was used as an internal control to normalize the expression levels of the genes tested. c, Defective S-phase entry of pre-BII cells from mCREB-1 Tg mice. Bone marrow cells (1 x 106/ml) from NTg and Tg mice were stimulated with media for 48 h. The cells were stained with CyChrome-conjugated anti-B220, PE-conjugated anti-CD43, and FITC-conjugated anti-CD24 Abs. After fixation and permeabilization with 1% paraformaldehyde and 70% ethanol, cells were stained with 2 µg/ml Hoechst 33343, followed by FACS analysis. The data were acquired in list-mode, and cell cycle analysis was performed using WinMDI Ver2.8 and Modfit LT Verity cell cycle analysis software. The data shown are a representative of three independent experiments.

 
JunB and c-Jun have been shown to play antagonistic roles in cell cycle regulation (40, 41). Both c-Jun and JunB have been implicated in G1-S phase transition (42, 43). Given that the pre-BII B cells in the normal bone marrow are actively proliferating cells, we tested whether the decrease in the pre-BII population is due to a block in G1 to S phase progression. The cell cycle profile of pre-BII B cells from Tg and their littermate NTg controls were tested in vitro. Consistent with previous observations, pre-BII B cells showed increased cycling status at 48 h in culture (Fig. 3c, top panel). However, cell cycle analysis of pre-BII cells from Tg mice exhibited a consistent decrease in S-phase entry compared with wild-type littermates (Fig. 3c (41 vs 11% in NTg and Tg mice, respectively). This is further reflected by accumulation of cells in G0-G1 and G2-M phase of the cell cycle.

Abnormal splenic B cell profiles in the mutant CREB-1 Tg mice

CREB-1 has been implicated to play a role in mature T and B cell signaling and development (22, 23, 25, 30, 33). To determine whether the abnormal B cell development is further reflected in the peripheral lymphoid organs, splenocytes, and lymph node cells from Tg and NTg mice were analyzed. When compared with NTg mice, the mCREB-1 Tg mice revealed ~40% decrease in the IgM+B220+ cells (29.3 ± 0.96% in NTg vs 17.7 ± 2.9% in Tg mice), with minimal change in CD4 or CD8 T cell population (Fig. 4a). This phenotypic distribution is further reflected in significant decrease in the absolute number of mature B cells in the spleen (36 ± 3.6 x 106 in NTg vs 22 ± 1.9 x 106 in Tg mice, p < 0.01). Interestingly, Tg mice revealed alterations in specific B cell populations in the spleen. Thus, when compared with NTg mice, mCREB-1 Tg mice revealed ~45% decrease in CD21dimCD23high follicular B cells with minimal change in the CD21highCD23dim marginal zone B cell (16.5 ± 1.4 x 106 in NTg vs 9.5 ± 0.92 x 106 cells in Tg mice p < 0.01 (Fig. 4c and Table II)).


Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 4. Decreased IgM+B220+ B cells associated with reduced follicular but not marginal zone B cells in mCREB-1 Tg mice. a, Splenocytes (top panel) and lymph node cells (bottom panel) from NTg and Tg littermates were stained with PE-conjugated anti-B220 and FITC-conjugated anti-IgM or PE-conjugated anti-CD4 and FITC-conjugated anti-CD8. The numbers represent the percentage of cells within the quadrants (left panel) or the boxes (right panel). The results shown are representative of four independent experiments with similar observations. b, Splenocytes from NTg and Tg littermate were stained with CyChrome-conjugated anti-B220 and biotinylated anti-CD23 and FITC-conjugated anti-CD21 followed by staining with PE-conjugated streptavidin. The expression levels of CD21 and CD23 of B220+ gated cells are shown. The numbers represent the percentage of cells in the B220+ population. The results shown are a representative of four independent experiments with similar outcomes. c, The absolute cell number of marginal zone (MZ) (CD21highCD23dim) and follicular (FO) (CD21dimCD23high) B cells from NTg and Tg mice are shown. The cells were identified as described in b and extrapolated with total splenocytes. Numbers represent mean ± SD from four independent experiments (*, p < 0.01; unpaired Student’s t test). d, Splenic B cells (2.5 x 105) from NTg or Tg mice were stimulated with medium, anti-IgM (10 µg/ml), or PMA (0.1 µg/ml) + ionomycin (0.5 µg/ml) in 200-µl volume for 40 h. The cells were then pulsed for another 8 h with 1 µCi of [3H]thymidine. The cells were harvested and the [3H]thymidine incorporation was measured as described in Materials and Methods. The data are represented as fold induction in response to activation compared with unstimulated conditions. Numbers represent the mean of fold induction ± SD from six independent experiments (*, p < 0.001; unpaired Student’s t test).

 

View this table:
[in this window]
[in a new window]
  		Table II. B cell populations in spleen of mCREB Tg micea

 
Defective humoral immune responses in mCREB-1 Tg mice

CREB/AP-1 family of proteins has been shown to regulate the expression of Ig gene (44). To test whether overexpression of dominant-negative CREB-1 in the mice alters the serum Ig, ELISA analysis of sera from 8-wk-old naive NTg and Tg mice was performed. Although minimal differences were observed in the levels of serum IgA, IgG, IgG2a, IgG2b, and IgG3, a consistent yet moderate increase (~30%) in total IgM levels in Tg mice compared with NTg littermates (38 ± 5 vs 29 ± 3 µg/ml, p < 0.001) (Fig. 5a).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Defective humoral immune response in the mCREB-1 Tg mice. a, Comparable serum Ab levels in Tg and NTg mice. Levels of IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs in preimmune sera from unimmunized NTg ({circ}) and mCREB-1 Tg mice (•) is shown. The results represent mean serum concentration of indicated isotype ± SD from 10 mice per group. b, Primary immune response to T-dependent Ag, DNP-KLH. NTg mice ({circ}) and mCREB-1 Tg mice (•) were injected with DNP-KLH (50 µg/mice) i.p. (IP). Seven days after immunization, the levels of TNP-KLH-specific IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs to the primary immune response were analyzed by ELISA as described in Materials and Methods. The results represent mean serum concentration of indicated isotype ± SD from 10 to 13 mice/group. c, Secondary immune response to T-dependent DNP-KLH. NTg ({circ}) and mCREB-1 Tg mice (•) were injected i.p. with DNP-KLH (50 µg/mice) 4 wk after the primary immunization. Five days after secondary immunization, the level of TNP-KLH-specific IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 Abs were analyzed by ELISA as described in Materials and Methods. The results represent mean serum concentration of indicated isotype ± SD from six to eight mice per group. d, Immune response to T-independent type 1(TNP-LPS) and type II (TNP-Ficoll) Ags. NTg ({circ}) and mCREB-1 Tg mice (•) were injected i.p. with TNP-LPS (50 µg/mice). Seven days after immunization, the levels of Ag-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 Abs were analyzed by isotype-specific ELISA as described in Materials and Methods. IgM response to T-independent type 2 Ag TNP-Ficoll (10 µg/ml i.p) is shown in the lower right most panel. The results represent mean serum concentration of indicated isotype ± SD from four to six mice per group.

 
To test the potential differences in immune responses in vivo, Tg, and NTg mice were immunized with DNP-KLH (T-dependent Ag), TNP-LPS (T-independent type 1 Ag), or TNP-Ficoll (T-independent type 2 Ag). Ag-specific (DNP/TNP-specific Ab response was evaluated in the preimmune, 7-day postprimary immune and in 5-day postsecondary immune sera. Tg and NTg mice exhibited comparable levels of anti-DNP-specific IgM, IgG2, IgG2b, and IgG3 levels during the primary immune response to DNP-KLH immunization. However, dramatic defects in secondary immune responses to DNP-KLH were observed in the Tg mice. Thus, in spite of the presence of comparable Ag-specific serum IgM levels, Tg mice exhibited significant reduction in Ag-specific IgG1 (5.7 ± 2.9 vs 2.2 ± 1.3 µg/ml (p < 0.005)), IgG2b (1.5 ± 0.66 vs 0.3 ± 0.16 µg/ml (p < 0.005)), and IgG3 (0.23 ± 0.06 vs 0.099 ± 0.06 µg/ml (p < 0.005)) Ab levels compared with age and sex matched controls (Fig. 5c). Thus, the expression of the IgG1, IgG2a, IgG2b, and IgG3 were reduced ~62, 91, 80, and 61%, respectively. Interestingly, a consistent increase in IgA levels during primary (~40%) (0.018 ± 0.005 vs 0.025 ± 0.008 µg/ml (p < 0.05)) and secondary (>100%) (0.011 ± 0.003 vs 0.024 ± 0.0069 µg/ml (p < 0.001)) responses in Tg mice compared with NTg mice (Fig. 5c). Furthermore, a significant decrease in IgG2a levels was noticed in the Tg mice in primary ((0.32 ± 0.14 vs 0.09 ± 0.05 µg/ml (p < 0.001)) and secondary IgG2a (1.6 ± 0.5 vs 0.4 ± 0.09 µg/ml (p < 0.001)) immune responses to T-dependent Ag (Fig. 5, b and c). In contrast to T-dependent Ag, Ag specific immune responses to T-independent TNP-LPS or TNP-Ficoll were comparable in the both Tg and NTg controls (Fig. 5d).

Abnormal accumulation of B-1 B cells that are resistant to apoptosis in mCREB-1 Tg mice

In contrast to the bone marrow and spleen, a consistent increase in total peritoneal cells was observed in Tg mice compared with NTg littermates (23.8 ± 5.2 x 105 and 37.9 ± 2.6 x 105 in NTg and Tg mice, (p < 0.01) (Table III)). Furthermore, flow cytometric analysis of peritoneal cells revealed significant increase in B220+IgM+ population in Tg mice compared with NTg (22 ± 3 and 33 ± 2.6% in NTg and Tg mice, p < 0.01; Table III). The increased cell number and the abnormal distribution reflected ~3-fold increase in IgM+B220+ peritoneal B cells in the Tg mice (13 ± 0.01 x 105) compared with the wild-type littermate (5 ± 0.01 x 105) controls. The IgM+B220+ cells were further confirmed to be B1 B cells based on the (IgMhighIgDlowMac-1+) staining (Fig. 6a). B1 B cells can be further divided into CD5+ B-1a and CD5– B1b cells based on the expression of CD5 surface marker. Flow cytometric analysis of peritoneal B cell with anti-B220-(PE-Cy5), anti-Mac-1-(PE), and anti-CD5-(FITC) showed ~3- to 4-fold increase in both B-1a (CD5+) and B-1b (CD5–) cells in Tg mice compared with NTg littermates (Fig. 6b and Table III).


View this table:
[in this window]
[in a new window]
  		Table III. B cell populations in peritoneum of mCREB Tg micea

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Abnormal accumulation of peritoneal B1a and B1b B cells in the mCREB-1 Tg mice. a, The peritoneal cells from NTg ({square}) and mCREB-1 Tg mice ({blacksquare}) were stained with CyChrome-conjugated anti-B220 and PE-conjugated anti-Mac-1. Numbers represent mean of B220+Mac-1+ B-1 cells ± SD from six mice. (*, p < 0.01; unpaired Student’s t test). b, The peritoneal cells from NTg ({square}) and mCREB-1 Tg mice ({blacksquare}) were stained with CyChrome-conjugated anti-B220, PE-conjugated anti-Mac-1, and FITC-conjugated anti-CD5. Numbers represent mean ± SD of B220+Mac-1+CD5+ B1a (left panel) or B220+Mac-1+CD5– B1b (right panel) cells from six mice. (*, p < 0.01; unpaired Student’s t test). c, The peritoneal cells from Tg mice are resistant to spontaneous apoptosis. The peritoneal cells (1 x 106) from NTg or Tg littermates were cultured for 24 h in the presence of media and stained with B220 (CyChrome), Annexin VFITC, and propidium iodide. The numbers in the upper right quadrant represent percentage of B220+Annexin V+ cells. Results from three independent experiments are shown in the bottom panel.

 
To determine whether the increased peritoneal B1 B cells is due to active proliferation and expansion, peritoneal cells were stained with CFSE and cultured in the presence or absence of LPS, anti-IgM, or PMA. Flow cytometric analysis of CFSE dye reduction in activated peritoneal B cells failed to reveal any differences in cell division over a period of 24–72 h tested (data not shown). To determine whether the increased B1 B cells in the mCREB Tg mice is due resistance to apoptosis, peritoneal cells from Tg and NTg mice were cultured in vitro for 24 h, and the apoptosis of B cells was determined by using B220/annexin V and propidium iodide staining. Peritoneal B220+ B cells from Tg mice showed 3- to 4-fold decrease in spontaneous apoptosis compared with the NTg littermates. (Fig. 6c).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The studies described in this report have identified differential role for CREB-1 in multiple stages of B cell development and functional maturation in vivo. Thus, overexpression of dominant -negative CREB-1 in B cells resulted in pre-BI to pre-BII B cell progression in the bone marrow, decreased follicular B cells in the spleen, increased B1 B cells in the peritoneum, and defective T-dependent Ab responses. The observed defects were noted in four independent lines, as well as in both FVB/N and (FVB/N x C57BL/6) mixed backgrounds (data not shown).

Significant reduction in mature, immature, and pre-BII B cells associated with an increase in pre-BI B cells suggests an early-stage-specific role for CREB-1 in pre-BI to pre-BII transition. The reduction in pre-BII cells in the mCREB-1 mice suggested a possible cell survival or proliferative defect in these cells. Prosurvival genes such as Bcl-2 or Bcl-xL are known to be involved in B cell development in the bone marrow (45, 46). Pre-BI to pre-BII transitional block in the mCREB-1 Tg mice is not due to deregulation of these antiapoptotic genes as analysis of sorted pre-BI and pre-BII B cells revealed comparable levels of Bcl-2 and Bcl-xL expression in Tg and NTg mice. Consistent with this, expression of Bcl-2 or Bcl-xL transgenes in the mCREB-1 Tg mice failed to rescue neither the accumulated pre-BI nor the reduced pre-BII phenotype in the double Tg mice (data not shown). The failure of Bcl-2 and Bcl-xL transgenes to rescue the defective pre-BII B cell expansion suggests possible involvement of other cell survival genes such as Mcl-1, XIAP, Bax, and Bad. Consistent with this hypothesis Mcl-1 gene promoter has been shown to be regulated by Ser133-phosphorylated CREB-1 in myeloid cell lines (47).

Several lines of evidences indicate a potential role for CREB-1 in the cell cycle regulation of pre-B cells. First, RT-PCR analysis of RNA from pre-BI and pre-BII B cells revealed the reciprocal regulation of JunB in pre-BI and pre-BII B cells. Thus, the increased JunB in pre-BII B cells can account for the decreased S-phase entry as reported previously (40, 41, 42, 43, 48). Consistent with this hypothesis, the mCREB-1 Tg pre-BII B cells exhibited defective cell cycle progression associated with decreased S-phase entry (Fig. 3c). It is likely that the defective B cell development found in the mCREB-1 Tg bone marrow could be attributed to the defective IL-7 signaling due to CREB-1-dependent deregulation of the AP-1 family members such as c-Fos and c-Jun (49, 50). Consistent with this, mice lacking IL-7, IL-7R{alpha}, or the common {gamma}c chain show a block of B cell development, which results in a reduction of pre-B cell populations (51, 52). Alternatively, a role for CREB-1 in IL-7-independent activation pathways involving stromal cell compartment cannot be ruled out. Comparable levels of expression of vpreB, {lambda}5, and mb-1 transcripts from sorted pre-BII cells indicate that the defective pre-BII development is less likely due to defective pre-BCR assembly, although a role for CREB-1 in pre-BCR-mediated signaling events remains to be tested. Nevertheless, the defective S-phase entry in mCREB-1 pre-BII B cells is consistent with a role for abnormally expressed JunB and c-Jun in the inhibition of cell cycle progression. Consistent with this hypothesis, overexpression of JunB has been shown to mediate an inhibitory effect on cell cycle progression through blocking cyclin D1, which in turn is required for the G1-S phase transition (40). The observed results emphasize the complex nature of gene regulation by AP-1 family members by CREB-1. For example, Jun family members exhibit different functional properties as transcription factors. Specifically, JunB is known to be a weak AP-1 transactivator as compared with c-Jun. Additional junB overexpression is known to antagonize some of the transcriptional activities of c-Jun (53, 54). Also, the effect of junB and c-jun depend on the promoter context, their phosphorylation status and cell type (55).

The observed developmental defects in the mCREB-1 Tg mice are selective for B but not T cells. The decrease in mature B cells in the spleen could be attributed to the developmental block in the bone marrow (56, 57). Alternatively, it is likely that the cells that had escaped the development block in the bone marrow could be still susceptible to CREB-1-dependent deregulation of cell proliferation. Consistent with this the mCREB-1 splenic B cells exhibited a moderate yet consistent decrease in proliferation in response to anti-IgM or PMA + ionomycin (Fig. 4d). This conclusion is further supported by a possible role for CREB-1 in the proliferation of mature B cells (24, 25, 33). The defective development of mature peripheral B cell population in the mCREB-1 Tg mice could be attributed to the change in BCR signaling strength, the specificity, and the repertoire usage in different immune compartments (58).

The defective secondary immune responses in the mCREB-1 Tg mice are intriguing. The selective decrease in Ag specific IgG1, IgG2a, IgG2b, and IgG3 Ab levels during secondary immune responses could be due to the defective responsiveness of Ag-specific B cells to T cell-derived cytokine. Consistent with this hypothesis, recently we demonstrated a critical role for CREB-1 in B cell responsiveness to IFN-{gamma} during AgR-mediated B cell activation (25). Alternatively, CREB-1 may regulate expression of distinct Ab isotypes. This is consistent with the CREB binding sites in several Ig promoters, including IgA and IgG1 (44, 59, 60).

The increased B1 B cells in the peritoneum can be attributed to several possibilities. The abnormal B1 B cell population may be attributed to the abnormal development of B1 B cells from the fetal liver precursor (61). Alternatively cells blocked in pre-BI stage in the bone marrow, could contribute to abnormal development, migration and/or subsequent development into B1 B cells in the peritoneum. Acquisition of B1 B cell surface phenotype by B2 B cells, through modulation of surface molecules upon Ag receptor engagement by multivalent cross-linking of surface IgM, has been observed in vitro (62). Two lines of evidences indicate that the increased B1 B cells in the peritoneal cavity are not due to abnormal proliferation. First, CFSE-labeled B1 B cells from Tg and NTg mice are indistinguishable in their ability to proliferate either in vitro or in vivo. Second, we failed to see differences in spontaneous or LPS-induced proliferation of B1 B cells from Tg and NTg littermate controls. Moreover, resistance to spontaneous or fludarabine-induced apoptosis by Tg peritoneal cells suggests potential regulation antiapoptotic genes by CREB-1 (47). Consistent with this hypothesis, accumulation of CD5+ B cells in chronic lymphocytic leukemia patients has been attributed to increased apoptosis mediated through up-regulation of expression of antiapoptotic genes such as Bcl-2 and Mcl-1. Thus, the increased resistance to apoptosis by B1 B-cells from Tg mice could be attributed to altered expression of any of the known or hitherto unknown antiapoptotic genes or their regulators. Consistent with this, CREB-1 has been shown to bind and regulate expression of the promoter regions of antiapoptotic genes such as Mcl-1 and Bcl-2 in vitro. In this context, it is less expected that dominant-negative CREB-1 in Tg mice rendered resistance to apoptosis in peritoneal B cells. The mechanistic basis for differential role of CREB-1 in B1 and B2 B cell is not clear. It is possible that phosphorylation events that are constitutively active in B1 but not B2 B cells, as evidenced by constitutively active STAT3, ERK, and NF-AT may attribute to dominant-negative CREB-1-dependent resistance to death (63, 64). It is likely that the ultimate CREB-1-dependent positive or negative physiological outcome on growth and viability of a cell type is dependent on the activation status of multiple signaling pathways, including protein kinase C, protein kinase A, CaMKinase, and Ras that regulate CREB-1 and its dimerization partners such as ATF-1. It is also likely the differential effect of CREB-1 in B1 and B2 B cells could be attributed to differential competition of phosphomutant CREB-1 to other CRE binding proteins such as ATF-1, ATF-2, or the cAMP response element modulator. Recently CREB-1 has been implicated to play a role in myeloid cell transformation (65, 66). Ongoing experiments aimed to identify natural CREB-1 targets in vivo using the mutant CREB-1 B cells will define the role for CREB-1 in B1 and B 2 B cell growth and survival and Ab responses.


   Acknowledgments
 
We thank Cindy McAllister for the help with the flow cytometry. Excellent secretarial support provided by Kathy Porter is greatly appreciated. We thank Drs. Carol Whitacre, Michael Caligiuri, and Azad Kaushik for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the American Cancer Society (to N.M.), Leukemia and Lymphoma Society of America (to J.C.B.), and The D. Warren Brown Foundation (to J.C.B. and N.M.). H.-C.C. is a recipient of a Raymon E. Mason Foundation Fellowship for graduate research. Back

2 Address correspondence and reprint requests to Dr. Natarajan Muthusamy, Chronic Lymphocytic Leukemia, Experimental Therapeutics Laboratory, Division of Hematology and Oncology, 1132C, James Cancer Hospital, 300 West 10th Avenue, Columbus, OH 43210. E-mail address: Muthusamy-1{at}medctr.osu.edu Back

3 Abbreviations used in this paper: ATF, activating transcription factor; CBP, CREB-binding protein; PCNA, proliferating cell nuclear Ag; Tg, transgenic; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl; NTg, nontransgenic; mCRE, mutant CRE oligonucleotide; HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication April 28, 2005. Accepted for publication October 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Montminy, M. R., K. A. Sevarino, J. A. Wagner, G. Mandel, R. H. Goodman. 1986. Identification of a cyclic-AMP responsive element within the rat somatostatin gene. Proc. Natl. Acad. Sci. USA 83: 6682-6686. [Abstract/Free Full Text]
  2. Comb, M., N. C. Burnberg, A. Seascholtz, E. Herbert, H. M. Goodman. 1986. A cyclic-AMP- and phorbol ester-inducible DNA element. Nature 323: 353-356. [Medline]
  3. Masquilier, D., P. Sassone-Corsi. 1992. Transcriptional cross-talk: nuclear factors CREM and CREB bind to AP-1 sites and inhibit activation by Jun. J. Biol. Chem. 267: 22460-22466. [Abstract/Free Full Text]
  4. Benbrook, D. M., N. C. Jones. 1990. Heterodimer formation between CREB and JUN proteins. Oncogene 5: 295-302. [Medline]
  5. Hai, T., T. Curran. 1991. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc. Natl. Acad. Sci. USA 88: 3720-3724. [Abstract/Free Full Text]
  6. Yamamoto, K. K., G. A. Gonzalez, W. H. Biggs, 3rd, M. R. Montminy. 1988. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334: 494-498. [Medline]
  7. Gonzalez, G. A., M. R. Montminy. 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59: 675-679. [Medline]
  8. Nichols, M., F. Weih, W. Schmid, C. DeVack, E. Kowenz-Leutz, B. Luckow, M. Boshart, G. Schutz. 1992. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. EMBO J. 11: 3337-3346. [Medline]
  9. Chrivia, J. C., R. P. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, R. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365: 855-859. [Medline]
  10. Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smeal, M. Karin, J. Feramisco, M. Montminy. 1994. Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370: 226-228. [Medline]
  11. Kwok, R. P., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. Roberts, M. R. Green, R. H. Goodman. 1994. Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370: 223-226. [Medline]
  12. Sun, P., R. A. Maurer. 1995. An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation. J. Biol. Chem. 270: 7041-7044. [Abstract/Free Full Text]
  13. Parker, D., K. Ferreri, T. Nakajima, V. J. LaMorte, R. Evans, S. C. Koerber, C. Hoeger, M. R. Montminy. 1996. Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism. Mol. Cell. Biol. 16: 694-703. [Abstract]
  14. Hagiwara, M., P. Brindle, A. Harootunian, R. Armstrong, J. Rivier, W. Vale, R. Tsien, M. R. Montminy. 1993. Coupling of hormonal stimulation and transcription via cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. Cell. Biol. 13: 4852-4859. [Abstract/Free Full Text]
  15. Xie, H., T. L. Rothstein. 1995. Protein kinase C mediates activation of nuclear cAMP response element- binding protein (CREB) in B lymphocytes stimulated through surface Ig. J. Immunol. 154: 1717-1723. [Abstract]
  16. Matthews, R. P., C. R. Guthrie, L. M. Wailes, X. Zhao, A. R. Means, G. S. McKnight. 1994. Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol. Cell. Biol. 14: 6107-6116. [Abstract/Free Full Text]
  17. Sheng, M., M. A. Thompson, M. E. Greenberg. 1991. CREB: a Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252: 1427-1430. [Abstract/Free Full Text]
  18. Sun, P., H. Enslen, P. S. Myung, R. A. Maurer. 1994. Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev. 8: 2527-2539. [Abstract/Free Full Text]
  19. Ginty, D. D., A. Bonni, M. E. Greenberg. 1994. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77: 713-725. [Medline]
  20. Bohm, M., G. Moellmann, E. Cheng, M. Alvarez-Franco, S. Wagner, P. Sassone-Corsi, R. Halaban. 1995. Identification of p90RSK as the probable CREB-Ser133 kinase in human melanocytes. Cell Growth Differ. 6: 291-302. [Abstract]
  21. Xing, J., D. D. Ginty, M. E. Greenberg. 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273: 959-963. [Abstract]
  22. Muthusamy, N., J. M. Leiden. 1998. A protein kinase C-, Ras-, and RSK2-dependent signal transduction pathway activates the cAMP-responsive element-binding protein transcription factor following T cell receptor engagement. J. Biol. Chem. 273: 22841-22847. [Abstract/Free Full Text]
  23. Xie, H., T. C. Chiles, T. L. Rothstein. 1993. Induction of CREB activity via the surface Ig receptor of B cells. J. Immunol. 151: 880-889. [Abstract]
  24. Xie, H., Z. Wang, T. L. Rothstein. 1996. Signaling pathways for antigen receptor-mediated induction of transcription factor CREB in B lymphocytes. Cell. Immunol. 169: 264-270. [Medline]
  25. Frissora, F., H. C. Chen, J. Durbin, S. Bondada, N. Muthusamy. 2003. IFN-{gamma}-mediated inhibition of antigen receptor-induced B cell proliferation and CREB-1 binding activity requires STAT-1 transcription factor. Eur. J. Immunol. 33: 907-912. [Medline]
  26. Dey, A., D. W. Nebert, K. Ozato. 1991. The AP-1 site and the cAMP- and serum response elements of the c-fos gene are constitutively occupied in vivo. DNA Cell Biol. 10: 537-544. [Medline]
  27. Amato, S. F., K. Nakajima, T. Hirano, T. C. Chiles. 1996. Transcriptional regulation of the junB promoter in mature B lymphocytes: activation through a cyclic adenosine 3',5'-monophosphate-like binding site. J. Immunol. 157: 146-155. [Abstract]
  28. Amato, S. F., K. Nakajima, T. Hirano, T. C. Chiles. 1997. Transcriptional regulation of the junB gene in B lymphocytes: role of protein kinase A and a membrane Ig-regulated protein phosphatase. J. Immunol. 159: 4676-4685. [Abstract]
  29. Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, M. B. Prystowsky. 1994. Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes. Mol. Cell. Biol. 14: 4233-4243. [Abstract/Free Full Text]
  30. Barton, K., N. Muthusamy, M. Chanyangam, C. Fischer, C. Clendenin, J. M. Leiden. 1996. Defective thymocyte proliferation and IL-2 production in transgenic mice expressing a dominant-negative form of CREB. Nature 379: 81-85. [Medline]
  31. Powell, J. D., C. G. Lerner, G. R. Ewoldt, R. H. Schwartz. 1999. The –180 site of the IL-2 promoter is the target of CREB/CREM binding in T cell anergy. J. Immunol. 163: 6631-6639. [Abstract/Free Full Text]
  32. Wilson, B. E., E. Mochon, L. M. Boxer. 1996. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis. Mol. Cell. Biol. 16: 5546-5556. [Abstract]
  33. Zhang, C. Y., Y. L. Wu, L. M. Boxer. 2002. Impaired proliferation and survival of activated B cells in transgenic mice that express a dominant-negative cAMP-response element-binding protein transcription factor in B cells. J. Biol. Chem. 277: 48359-48365. [Abstract/Free Full Text]
  34. Rudolph, D., A. Tafuri, P. Gass, G. J. Hammerling, B. Arnold, G. Schutz. 1998. Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein. Proc. Natl. Acad. Sci. USA 95: 4481-4486. [Abstract/Free Full Text]
  35. Hummler, E., T. J. Cole, J. A. Blendy, R. Ganss, A. Aguzzi, W. Schmid, F. Beermann, G. Schutz. 1994. Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Proc. Natl. Acad. Sci. USA 91: 5647-5651. [Abstract/Free Full Text]
  36. Su, G. H., H. M. Chen, N. Muthusamy, L. A. Garrett-Sinha, D. Baunoch, D. G. Tenen, M. C. Simon. 1997. Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B. EMBO J. 16: 7118-7129. [Medline]
  37. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, et al 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68: 855-867. [Medline]
  38. Rolink, A. G., J. Andersson, F. Melchers. 2004. Molecular mechanisms guiding late stages of B-cell development. Immunol. Rev. 197: 41-50. [Medline]
  39. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213-1225. [Abstract/Free Full Text]
  40. Bakiri, L., D. Lallemand, E. Bossy-Wetzel, M. Yaniv. 2000. Cell cycle-dependent variations in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. EMBO J. 19: 2056-2068. [Medline]
  41. Mechta-Grigoriou, F., D. Gerald, M. Yaniv. 2001. The mammalian Jun proteins: redundancy and specificity. Oncogene 20: 2378-2389. [Medline]
  42. Shaulian, E., M. Karin. 2001. AP-1 in cell proliferation and survival. Oncogene 20: 2390-2400. [Medline]
  43. Shaulian, E., M. Karin. 2002. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4: E131-E136. [Medline]
  44. Bhushan, A., L. R. Covey. 2001. CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Ig {gamma}1 proximal promoter. Eur. J. Immunol. 31: 653-664. [Medline]
  45. Strasser, A., A. W. Harris, L. M. Corcoran, S. Cory. 1994. Bcl-2 expression promotes B- but not T-lymphoid development in scid mice. Nature 368: 457-460. [Medline]
  46. Fang, W., D. L. Mueller, C. A. Pennell, J. J. Rivard, Y. S. Li, R. R. Hardy, M. S. Schlissel, T. W. Behrens. 1996. Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. Immunity 4: 291-299. [Medline]
  47. Wang, J. M., J. R. Chao, W. Chen, M. L. Kuo, J. J. Yen, H. F. Yang-Yen. 1999. The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol. Cell. Biol. 19: 6195-6206. [Abstract/Free Full Text]
  48. de Groot, R. P., P. Sassone-Corsi. 1992. Activation of Jun/AP-1 by protein kinase A. Oncogene 7: 2281-2286. [Medline]
  49. Fujita, K., S. Imoto, J. Phuchareon, T. Tokuhisa. 1994. c-fos and jun gene expression in murine precursor B lymphocytes developed in the interleukin-7-dependent bone marrow cell culture. Immunobiology 190: 13-22. [Medline]
  50. Imoto, S., L. Hu, Y. Tomita, J. Phuchareon, U. Ruther, T. Tokuhisa. 1996. A regulatory role of c-Fos in the development of precursor B lymphocytes mediated by interleukin-7. Cell. Immunol. 169: 67-74. [Medline]
  51. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526. [Abstract/Free Full Text]
  52. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
  53. Schütte, J., J. Viallet, M. Nau, S. Segal, J. Fedorko, J. Minna. 1989. jun-B inhibits and c-fos stimulates the transforming and trans-activating activities of c-jun. Cell 59: 987-997. [Medline]
  54. Deng, T., M. Karin. 1993. JunB differs from c-Jun in its DNA-binding and dimerization domains, and represses c-Jun by formation of inactive heterodimers. Genes Dev. 7: 479-490. [Abstract/Free Full Text]
  55. Mauviel, A., K. Y. Chung, A. Agarwal, K. Tamai, J. Uitto. 1996. Cell-specific induction of distinct oncogenes of the Jun family is responsible for differential regulation of collagenase gene expression by transforming growth factor beta in fibroblasts and keratinocytes. J. Biol. Chem. 271: 10917-109223. [Abstract/Free Full Text]
  56. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morony, A. J. Oliveira-dos-Santos, G. Van, A. Itie, et al 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: 315-323. [Medline]
  57. Manabe, N., H. Kawaguchi, H. Chikuda, C. Miyaura, M. Inada, R. Nagai, Y. Nabeshima, K. Nakamura, A. M. Sinclair, R. H. Scheuermann, M. Kuro-o. 2001. Connection between B lymphocyte and osteoclast differentiation pathways. J. Immunol. 167: 2625-2631. [Abstract/Free Full Text]
  58. Cariappa, A., S. Pillai. 2002. Antigen-dependent B-cell development. Curr. Opin. Immunol. 14: 241-249. [Medline]
  59. Mao, C. S., J. Stavnezer. 2001. Differential regulation of mouse germline Ig {gamma}1 and {epsilon} promoters by IL-4 and CD40. J. Immunol. 167: 1522-1534. [Abstract/Free Full Text]
  60. Shi, M. J., S. R. Park, P. H. Kim, J. Stavnezer. 2001. Roles of Ets proteins, NF-{kappa}B and nocodazole in regulating induction of transcription of mouse germline Ig{alpha} RNA by transforming growth factor beta1. Int. Immunol. 13: 733-746. [Abstract/Free Full Text]
  61. Thomas-Vaslin, V., A. Coutinho, F. Huetz. 1992. Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells. Eur. J. Immunol. 22: 1243-1251. [Medline]
  62. Hardy, R. R., K. Hayakawa. 1991. A developmental switch in B lymphopoiesis. Proc. Natl. Acad. Sci. USA 88: 11550-11554. [Abstract/Free Full Text]
  63. Karras, J. G., Z. Wang, L. Huo, R. G. Howard, D. A. Frank, T. L. Rothstein. 1997. Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes. J. Exp. Med. 185: 1035-1042. [Abstract/Free Full Text]
  64. Wong, S. C., W. K. Chew, J. E. Tan, A. J. Melendez, F. Francis, K. P. Lam. 2002. Peritoneal CD5+ B-1 cells have signaling properties similar to tolerant B cells. J. Biol. Chem. 277: 30707-30715. [Abstract/Free Full Text]
  65. Gubina, E., X. Luo, E. Kwon, K. Sakamoto, Y. F. Shi, R. A. Mufson. 2001. beta Cytokine receptor-induced stimulation of cAMP response element binding protein phosphorylation requires protein kinase C in myeloid cells: a novel cytokine signal transduction cascade. J. Immunol. 167: 4303-4310. [Abstract/Free Full Text]
  66. Shankar, D. B., J. C. Cheng, K. Kinjo, N. Federman, T. B. Moore, A. Gill, N. P. Rao, E. M. Landaw, K. M. Sakamoto. 2005. The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia. Cancer Cell 7: 351-354. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Gururajan, A. Simmons, T. Dasu, B. T. Spear, C. Calulot, D. A. Robertson, D. L. Wiest, J. G. Monroe, and S. Bondada
Early Growth Response Genes Regulate B Cell Development, Proliferation, and Immune Response
J. Immunol., October 1, 2008; 181(7): 4590 - 4602.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.-C.
Right arrow Articles by Muthusamy, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.-C.
Right arrow Articles by Muthusamy, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS