![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, October 2005, p. 8531-8540, Vol. 25, No. 19
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.19.8531-8540.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Xiaozhong Shi,1,¶
Arianna Caprioli,1,¶
Hong-Hsing Liu,1,¶
Claudia Waskow,4
Keng-Mean Lin,3
Toru Miyazaki,2
Hans-Reimer Rodewald,4 and
Thomas N. Sato1,5*
Departments of Internal Medicine and Molecular Biology,1 Center for Immunology,2 Alliance for Cellular Signaling and Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390,3 Department of Immunology, University of Ulm, D-89070 Ulm, Germany,4 Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 100215
Received 4 December 2005/ Returned for modification 27 January 2005/ Accepted 7 July 2005
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In contrast to these in vitro data, very little is known about the physiological functions of BTB-kelch proteins in vivo, particularly in mammals. The most extensively studied mammalian BTB-kelch is Keap1 (12). Keap1 interacts with the transcription factor Nrf2, which regulates the expression of downstream genes encoding detoxifying and antioxidant proteins (12). Deletion of the Keap1 gene in mice results in the constitutive activation of Nrf2 and postnatal lethality (30). Another mammalian BTB-kelch protein whose in vivo function has been studied is Kelch homolog 10 (KLHL10) (33). Haploinsufficiency of this gene in mice causes infertility (33). The third mammalian BTB-kelch gene known to have an important physiological function is gigaxonin, which is mutated in giant axon neuropathy (2, 29). Our bioinformatics analysis of the human and mouse genomes has identified at least 38 and 42 BTB-kelch proteins, respectively (H.-H. Liu and T. N. Sato, unpublished). The function of most of these BTB-kelch proteins is unknown.
We have isolated a BTB-kelch protein, KLHL6, by virtue of its expression in embryonic but not adult endothelial cells (see below). The same gene was recently shown to be highly expressed in sheep Peyer's patch and human tonsil B cells (9). Based on this specific expression pattern in adult mice, it has been suggested that KLHL6 might be involved in B-cell functions, notably the germinal center reaction (9). Here, we describe B-cell compartments and B-cell functions in constitutive and conditional KLHL6-deficient mice. While early stages of B-cell development were unaffected, the loss of KLHL6 expression leads to reduced numbers of mature B cells. Antigen-dependent germinal center formation and B-cell antigen receptor (BCR) signaling were impaired in mice lacking KLHL6. Thus, in this report we establish a role for a BTB-Kelch protein in BCR signal transduction and germinal center formation.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Full-length cDNA was isolated by screening the mouse spleen lambda ZAP II library (Stratagene) using the 0.7-kb cDNA fragment cloned from the subtracted cDNA library. The analysis of the full-length cDNA revealed that this gene encoded a novel BTB-kelch protein (see Fig. S1A and B in the supplemental material).
Generation of constitutive KLHL6–/– mice. The bacterial artificial chromosome (BAC) genomic clone for the KLHL6 gene was obtained from Research Genetics and the targeting vector was constructed as shown in Fig. S1D in the supplemental material. Electroporation, embryonic stem (ES) cell culture, and Southern blot screening were performed as previously described (28). Chimeric mice were crossed with the CAG:Cre line to remove PGKneo (23). Mice were kept for further studies in CD1, 129/svEms-+Ter?/J, and BALB/c backgrounds. The mouse genotype was analyzed by PCR on genomic DNA using the primers indicated in Fig. S1D in the supplemental material.
Conditional gene targeting of KLHL6. The first exon of the KLHL6 gene was replaced by a fragment containing a floxed first exon followed by an Flp recombination target-flanked PGKneo cassette (see Fig. S3A in the supplemental material). The PGKneo cassette was removed by crossing chimeric mice with hACTB::FLPe.9205 mice (22).
Genetic background and age of the mice. The mice were studied on CD1, 129/svEms-+Ter?/J, and BALB/c backgrounds and the same phenotype was found in all of these backgrounds. The data presented in the manuscript were obtained from the CD1 background mice. The studies were conducted with adult mice at 6 to 10 weeks of age.
Flow cytometry. Splenocytes were isolated by mechanically dissociating mouse spleens through 100-µm nylon mesh (BD Bioscience) in the FACS staining buffer (5% bovine serum albumin-phosphate-buffered saline). Bone marrow cells were collected from femurs and tibias. Antibodies against B220 (RA3-6B2), CD21 (7G6), BP-1 (6C3), CD24 (M1/69), CD43 (S7), CD23 (B3B4), CD40, CXCR5, CD69, and immunoglobulin M (IgM) (II/41) coupled to allophycocyanin, phycoerythrin, fluorescein isothiocyanate, or biotin as appropriate were from BD Bioscience. Streptavidin coupled to peridinin chlorophyll protein (BD Bioscience) was used as the secondary reagent. At least 5 x 105 total cells were analyzed for each experiment. Cells were stained on ice in FACS buffer for 15 min and washed once before analyzing or adding the secondary reagent. In the latter case, cells were stained for another 15 min on ice followed by another washing step. Cells were detected by FACSCalibur or FACScan (Becton Dickinson) flow cytometers and analyzed using CellQuest software.
Footpad assay. Ovalbumin (50 µg/50 µl in PBS) together with 50 µl complete Freund's adjuvant was injected in one footpad of each mouse. The other footpad was injected with PBS as a negative control. Mice were anesthetized 10 days after the injection, and perfused with 4% paraformaldehyde-PBS. Popliteal lymph nodes were dissected, fixed for 4 h at 4°C in 4% paraformaldehyde-PBS, incubated overnight in 18% sucrose at 4°C, and embedded in OCT. Sections (8 µm) were blocked in 3% bovine serum albumin-PBS in room temperature for 1 h and incubated overnight at 4°C with 0.1 µg/ml peanut fluorescein isothiocyanate (FITC)-lectin (Sigma L-7381) in 3% bovine serum albumin-PBS. This lectin staining identifies germinal center B cells in lymph nodes (4). The stained samples were washed, mounted in VECTASHIELD (Vector Laboratories), and examined with an Axioplan microscope (Zeiss) equipped with fluorescence filters. For the analysis of KLHL6 expression in lymph nodes, popliteal lymph nodes were fixed in 4% paraformaldehyde-PBS and paraffin sections were prepared. In situ hybridization was carried out as previously described (24). Germinal center B cells in lymph nodes were measured by staining with antibodies GL7 (BD Bioscience) together with anti-B220.
ELISA for the detection of antiovalbumin antibodies. Each mouse was injected intraperitoneally with 10 µg ovalbumin dissolved in 100 µl PBS together with 100 µl CFA as an emulsion. After 7 and 14 days, mice were boosted with 10 µg ovalbumin dissolved in 100 µl PBS together with 100 µl incomplete Freund's adjuvant. Sera were collected from the mice before the primary ovalbumin injection, 6 days after the first boost, and 6 days after the second boost. Enzyme-linked immunosorbent assay (ELISA) plates were coated overnight at 4°C with 5 µg/ml ovalbumin in 0.05 M carbonate buffer, pH 9.6. On the next day, plates were blocked in 1% bovine serum albumin-PBS, washed three times with PBS-0.05%Tween 20, and loaded with 100 µl of diluted sera (dilutions of 1:10, 1:100, 1:1,000, and 1:10,000 in PBS, in triplicate) and incubated overnight at 4°C. On the following day, the plates were washed three times and incubated for 1 hour at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (dilution, 1:3,000). Each well was then washed three times, followed by colorimetric assay with a peroxidase substrate kit (Bio-Rad). Color development was stopped with 2% oxalic acid and the plates were analyzed by a plate reader (405 nm). The optical density at 405 nm was in the linear range between 0 and 1 with the sera diluted to 1:10,000, and these values were used to represent the titers.
In vitro cell proliferation assay. Spleens were removed from the mice and splenocytes were isolated by mechanical dissociation. B cells were purified by depletion of non-B cells using the mouse B-cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. The purity of the resulting B-cell fraction (>99%) was confirmed by FACS with anti-B220-phycoerythrin and anti-IgM-FITC antibodies. Approximately 106 cells per 96-well plate were seeded in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, antibiotics, and ß-mercaptoethanol. One hour later, the cells were stimulated with lipopolysaccharide, anti-CD40, and anti-IgM for 48 h. Following the stimulation, the cells were incubated with 0.5 µCi [3H]thymidine per well for an additional 20 h. The labeled cells were collected with a cell harvester and the [3H]thymidine incorporation was quantified by liquid scintillation counting.
RT-PCR analysis of KLHL6 expression in B-lymphocyte subsets. B cells were sorted according to the cell surface phenotypes reported by Hardy et al. and by Carsetti and colleagues (5, 11). The cDNAs were synthesized from total RNA isolated from approximately 20,000 cells from each indicated developmental stage. The primers used for KLHL6 were 5'-AGA CCA GGA GAG TGT GCA TGG-3' and 5'-CGT TGA TGG CAG GAC AAA GAG-3'. The primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Clontech. PCR was performed at 94°C (5 minutes), 35 cycles at 94°C (30 seconds), 58°C (30 seconds) 72°C (45 seconds), and 72°C (10 minutes) in the end.
Assays of intracellular free calcium levels in B lymphocytes. Calcium concentrations were measured by the protocol (PP00000011) of the Alliance for Cellular Signaling (www.signaling-gateway.org). B cells isolated by a B-cell isolation kit (Miltenyi Biotec 130-090-862) were resuspended at 5 x 106 cells/ml in SIMDM (Iscove’s modified Dulbecco’s medium supplied with 2 mM L-glutamine, 55 µM 2-mercaptoethanol, 0.025% Pluronic F-68, and 0.1 mg/ml bovine serum albumin) containing 12 µl/ml Fluo-3 (4 µM) (Molecular Probes) and 12 µl/ml Pluronic F-127 (0.08%) (Molecular Probes). Cells were distributed into six-well ultra-low-attachment plates (Corning) at 2 ml/well. Incubated at 37°C and 5% CO2 for 30 min, cells were resuspended in fresh SIMDM for another 30 min, pelleted down and resuspended in Hanks' balanced salt solution-bovine serum albumin (Hanks' balanced salt solution supplied with 25 mM HEPES, 1 mg/ml bovine serum albumin, pH 7.4) at 8.3 x 106 cells/ml. The final cell suspension was distributed into 96-well black-walled plates at 90 µl/well and equilibrated in the fluorescence chamber for 5 minutes
Baseline fluorescence was measured for 10 minutes. Ten µl of the F(ab')2 fragment of goat anti-mouse IgM or anti-CD19 was added to start the stimulation. The fluorescence was measured by Fluoroskan Ascent microplate fluorometer (Thermo-Labsystems) and analyzed by Ascent software (Thermo-Labsystems). For measurements of intracellular calcium levels by FACS, splenocytes were washed with 10% fetal bovine serum-PBS, resuspended at 107 cells/ml in loading medium (10% fetal bovine serum-RPMI 1640), and incubated with 5 µg/ml Indo-1 AM (Molecular Probes) for 30 min at 37°C. The cells were stained with phycoerythrin-conjugated anti-B220 monoclonal antibody for 30 min on ice, washed with Hanks' balanced salt solution twice, and resuspended in loading buffer at 107 cells/ml. Calcium flux was triggered by adding a F(ab')2 fragment of goat anti-mouse IgM (Jackson ImmunoResearch Laboratories) or anti-CD19 antibody (MB19-1, BD Biosciences). The calcium fluxes were recorded in real time using a FACSVantage SE instrument (BD Biosciences) and analyzed by FACSDiva (BD Biosciences).
Inositol 1,4,5-trisphosphate (IP3) measurement. B cells were resuspended at 2 x 107 cells/ml in RPMI 1640 (supplied with 25 mM HEPES) at 37°C for 30 min. B-cell stimulation was initiated by adding a 100-µl cell suspension into a tube with a 5-µg F(ab')2 fragment of goat anti-mouse IgM, and incubated at 37°C for the indicated periods. The reaction was stopped by adding 100 µl 10% perchloric acid, and the IP3 level was measured by the D-myoinositol 1,4,5-triphosphate (IP3) [3H] Biotak assay system (Amersham) according to the manufacturer's protocol.
Immunoprecipitation and immunoblotting. B cells were resuspended in RPMI 1640 (supplied with 25 mM HEPES) at 2 x 107 cells/ml. F(ab')2 fragments of goat anti-mouse IgM were diluted in RPMI 1640 at 30 µg/ml. The cell suspension and the diluted antibody solution were warmed at 37°C for 2 minutes prior to the stimulation. The stimulation was initiated by adding 500 µl of diluted antibody into the 500 µl B-cell suspension (total, 107 cells), and incubated at 37°C for the indicated periods. Cells were collected by centrifugation, and lysed using 200 µl ice-cold lysis buffer (Phosphosafe extraction buffer from Novagen) containing the complete protease inhibitor cocktail (Roche), Pefabloc SC (Roche), and 1 mM dithiothreitol on ice for 30 min. Cell lysate (2 x 107 cells for Btk, 107 for all the others) was precleared by protein G-agarose (Roche), incubated with the indicated antibody at 4°C for a period ranging from 4 h to overnight, and incubated with protein G-agarose for another 3 h. The protein G-agarose was washed with lysis buffer 3 times, and finally resuspended in loading buffer.
The immunoprecipitated products were heated and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blot analyses. ECL plus (Amersham) was used to detect the signals, and each band was quantified by Storm 860 (Molecular Dynamics). Data were analyzed by ImageQuant software (Molecular Dynamics). The blots were stripped for reprobing with IgG elution buffer (Pierce) according to the user's manual. Anti-phospholipase C2 (PLC2)(Q-20), anti-Syk(C-20), anti-Syk(N-19), and antiphosphotyrosine antibody (PY99) were purchased from Santa Cruz. Anti-Btk polyclonal antibodies were generously provided by Anne Satterthwaite and Owen Witte.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Others have recently reported that KLHL6 is expressed predominantly in sheep Peyer's patch and human tonsil B lymphocytes (9). To extend this analysis more systematically to various stages of B-cell ontogeny, B-cell progenitors were purified from the bone marrow of normal mice according to Hardy et al. (10). B cells develop from fraction B (B220+ CD43+ CD24+ BP-1–), via C (B220+ CD43+ CD24+ BP-1+), D (B220+ CD43– IgM–), and E (B220+ CD43– IgM+) to F (B220high CD43– IgM+). In this order, B to E represent immature and newly generated B cells, while cells in F represent recirculating mature B cells in the bone marrow.
Reverse transcription-PCR (RT-PCR) analysis showed similar expression of KLHL6 throughout B-cell development (Fig. 1A). Splenic B cells were sorted into transitional 1 (T1) (B220+ HSAhigh CD21– CD23–), T2 (B220+ HSAlow CD21+ CD23+), marginal zone (Mz) (B220+ CD21+ CD23–), and follicular (Fo) (B220+ HSA–/low CD21low CD23low) B-cell subsets (6, 16). Again, all of these B-cell populations expressed KLHL6 (Fig. 1A).
|
|
Next, we determined the stage in B-cell development at which loss of KLHL6 might affect B-cell development. Frequencies of pro- and pre-B-cell phenotypes in bone marrow were compared between KLHL6–/– and heterozygous littermates (Table 2). No differences were observed for fractions A to D. However, the frequency of cells in fraction E (immature) was increased by about 50%, and the mature population (fraction F) was decreased two- to threefold (Table 2; see also Fig. S2 in the supplemental material). These results indicate that KLHL6 was not required for the early stages of B-cell development. However, loss of KLHL6 affected fractions E and F.
|
Upregulation of KLHL6 expression during germinal center formation, and impaired germinal center formation in mice lacking KLHL6. It has previously been reported that KLHL6 is expressed in sheep Peyer's patch and human tonsil B cells, suggesting that KLHL6 expression may be associated with the germinal center (GC) reaction (9). To test this idea directly, mice were immunized in the footpad with ovalbumin. Wild-type and heterozygous mice exhibited visibly enlarged local lymph nodes. Interestingly, in situ hybridization demonstrated a strong upregulation of KLHL6 expression in the GC (Fig. 2A, left). Thus, KLHL6 expression increases upon antigen-driven GC formation in vivo.
|
To determine whether these morphological differences in GC formation correlated with functional alterations, we measured the ovalbumin-specific immunoglobulin G titers before and after primary and secondary immunization with ovalbumin (Fig. 2C). While the primary antibody response was comparable in both wild-type and KLHL6–/– mice, the memory IgG response was clearly reduced in the mutant (Fig. 2C). These data represent the first indication that KLHL6 is functionally important, albeit not essential, for GC formation.
B-lymphocyte-autonomous role of KLHL6 in germinal center formation. The analyses of the expression pattern of KLHL6, and the data on KLHL6–/– mice suggested that KLHL6 plays a role in B cells. However, it remained possible that the function of KLHL6 in non-B lymphocytes indirectly regulated B-lymphocyte development or function. In order to address this issue, we generated and studied a mouse line in which the KLHL6 gene was specifically deleted in B cells. To generate a conditional null allele of KLHL6, the first exon of the KLHL6 gene was flanked by a pair of loxP elements in ES cells (see Fig. S3A in the supplemental material). Mutant ES cells were used to establish the "floxed" KLHL6e1+/loxP mouse line (see Fig. S3B in the supplemental material) which was crossed to the CD19::Cre line. The latter has previously been shown to induce B-cell-specific gene deletion in vivo (21). Southern blot analyses of DNA from appropriate F1 mice demonstrated efficient and specific excision of the first exon of KLHL6 in B but not T cells (see Fig. S3C in the supplemental material).
To distinguish between a B-cell-intrinsic versus a B-cell-extrinsic effect of the loss of KLHL6, B-cell populations and functions were compared in B-cell-specific KLHL6 gene deletion mice, termed KLHL6e1loxP/loxP CD19::Cre, KLHL6+/– and KLHL6e1loxP/loxP control mice. Flow cytometric analyses of bone marrow and spleen showed comparable alterations in B-cell subsets in the KLHL6e1loxP/loxP CD19::Cre mouse line compared to the KLHL6–/– control mice (data not shown). These results indicate that KLHL6 has a cell-autonomous function in B lymphocytes in vivo.
Next, KLHL6e1loxP/loxP CD19::Cre, KLHL6+/– and KLHL6e1loxP/loxP control mice were immunized by ovalbumin injections. In agreement with the data from the constitutive KLHL6–/– mouse, PNA staining revealed compromised GC formation in the lymph nodes of KLHL6e1loxP/loxP CD19::Cre mice (Fig. 3). Quantification by FACS revealed a 228-fold versus 19-fold increase of B220+ GL7+ GC B lymphocytes per lymph node in response to ovalbumin injections in KLHL6e1loxP/lox and KLHL6e1loxP/loxP CD19::Cre mice, respectively. These experiments demonstrate a cell-intrinsic requirement for KLHL6 expression in B cells.
|
|
|
2 and Btk tyrosine kinase, but not of Syk tyrosine kinase (Fig. 5D to I). These biochemical analyses suggested that KLHL6 was involved in antigen-stimulated B-cell activation via Btk-mediated BCR signaling pathways. However, compared to the reduction in B-cell proliferation, the measurable defects in BCR signaling were rather mild. |
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 activation in vitro. Finally, we demonstrate a B-cell-autonomous function of KLHL6. The most striking aspect of the mutant phenotype reported here was the failure of KLHL6–/– mice to mount a full GC reaction in vivo. In a previous screen for genes differentially expressed in B cells undergoing immunoglobulin hypermutation, two genes have emerged as interesting candidates: the RING finger ubiquitin ligase Deltex-1, and KLHL6 (9). A role for Deltex-1 in the immune system has recently been excluded based on a detailed analysis of Deltex-1-deficient mice (27). With regard to KLHL6, expression of this gene in lymphoid tissues (9), specifically in all stages of B-cell development (Fig. 1), suggested a role in B cells. Moreover, expression of KLHL6 in sheep Peyer's patch and human tonsil B cells (9), together with the fact that KLHL6 expression was a strong histological marker of the GC (Fig. 2), suggested a functional relevance of KLHL6 expression for GC B cells. However, until now, definitive information on the role of KLHL6 has been lacking because mutant mice lacking this gene have not been reported.
The in vivo data on the GC phenotype and the B-cell numbers correlated with the impaired B-lymphocyte proliferation and compromised calcium flux in response to anti-IgM stimulation in vitro. The flow cytometric calcium flux analysis was done by gating on CD19+ B cells. Therefore, we cannot draw conclusions regarding the ability of rare B-cell subpopulations such as transitional or marginal zone B cells to show calcium responses.
Continuous expression of a fully signaling competent BCR is essential for the maintenance of the peripheral B-lymphocyte pool (14). Hence, the involvement of KLHL6 in BCR signaling could be important for antigen-stimulated B-lymphocyte proliferation. Along this line, in addition to GC B cells, the numbers of other B-cell subsets (T1, T2, and follicular B cells) were also quantitatively affected by the loss of KLHL6, suggesting a role of this gene in the generation and/or maintenance of normal B-cell compartments in vivo.
Although we do not have data to pinpoint exactly how KLHL6 is involved in the BCR signaling, a few possibilities can be proposed. It is possible that KLHL6 can serve as an adaptor for cullin 3 ubiquitin ligases for a putative substrate that affects BCR signaling. Another possibility is that KLHL6 is involved in regulating the cytoskeletal remodeling of B lymphocytes. It has been shown that many BTB-kelch proteins are important for normal cytoskeletal organization and remodeling (1). The normal cytoskeletal organization/remodeling and its related signaling and cell biological events are important for PLC2 signaling (3, 17). Thus, KLHL6 may be important for cytoskeletal organization, and deficiency of this KLHL6 function could result in impaired PLC2 signaling. It is conceivable that these aspects of impaired BCR signaling contribute to the underlying defects in B-cell function. However, the differences in Ca2+ influx, IP3 production, and the activation of Btk and PLC2 between the wild-type and KLHL6–/– B lymphocytes were marginal, albeit statistically significant. In contrast, the proliferation of B lymphocytes was severely compromised. This discrepancy may indicate that the observed alterations in BCR signal transduction do not fully reflect the key mechanism underlying the role of KLHL6 in B cells. The exact mechanism(s) remains to be determined.
KLHL6 is the third member of mammalian BTB-kelch proteins whose physiological function has been directly demonstrated. The other two are Keap1 and KLHL10 (30, 33). These studies showed rather diverse physiological functions of BTB-kelch proteins. Is there a unifying molecular and biochemical mechanism underlying these diverse functions of BTB-kelch proteins? Some of the BTB-kelch proteins, including Keap1, are associated with actin in cells (1). We generated an epitope-tagged KLHL6 protein which we expressed in cultured cells. This protein was mostly localized in the perinuclear region, but no consistent colocalization with actin was detected (data not shown). A new protein motif, the BACK (for BTB and C-terminal Kelch) domain, is present in most BTB-kelch proteins (26). Our in silico analysis of Keap1, KLHL10, and KLHL6 sequences showed that they all contain the BACK domain. However, the relevance of this novel domain to the apparent diverse physiological functions among BTB-kelch proteins remains unknown.
Our results show that KLHL6 is not essential for GC formation but that KLHL6 deficiency results in impaired GC formation in peripheral lymph nodes (Fig. 2 and 3). This GC phenotype may be a consequence of the lowered threshold of antigen-driven BCR signaling leading to reduced B-lymphocyte proliferation in the GC. However, it is also possible that KLHL6 possesses a yet-unidentified function in B cells that is crucial for normal GC formation. We have partially tested this possibility by analyses of expression of CD40 and CXCR5, known to play a critical role in GC formation (see Fig. S5 in the supplemental material). This analysis showed a normal level of expression of CD40 and CXCR5 in KLHL6–/– B lymphocytes, suggesting that the functional role of KLHL6 in GC formation occurs independently of CD40 or CXCR5.
Collectively, this first report has demonstrated a role for KLHL6 in GCs but has also raised many other questions which shall be addressed in detailed future experiments. Because immunoglobulin isotype switching and somatic hypermutation of immunoglobulin hypervariable regions represent important immunological events in GCs, it should be interesting to compare wild-type and KLHL6–/– mice for their capacity to mount these responses.
|
ACKNOWLEDGMENTS |
|---|
J.K. was supported in part by Deutsche Forschungsgemeinschaft (KR1887/2-1). This work was initially funded by the Sponsored Research Agreement with Pharmacia and later supported by the NIH (T.N.S.). H.-R.R. was supported by DFG (SFB-497-B5).
We declare that we have no competing financial interests.
|
FOOTNOTES |
|---|
Supplemental material for this article may be found at http://mcb.asm.org.
¶ These four authors contributed equally to this work.
Present address: Tumor Biology Center, Freiburg i.Br., Germany.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, P. Landrieu, F. Hentati, and M. Koenig. 2000. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat. Genet. 26:370-374.[CrossRef][Medline]
3. Bunnell, S. C., V. Kapoor, R. P. Trible, W. Zhang, and L. E. Samelson. 2001. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14:315-329.[CrossRef][Medline]
4. Butcher, E. C., R. V. Rouse, R. L. Coffman, C. N. Nottenburg, R. R. Hardy, and I. L. Weissman. 1982. Surface phenotype of Peyer's patch germinal center cells: implications for the role of germinal centers in B-cell differentiation. J. Immunol. 129:2698-2707.[Abstract]
5. Carsetti, R. 2004. Characterization of B-cell maturation in the peripheral immune system. Methods Mol. Biol. 271:25-35.[Medline]
6. Carsetti, R., M. M. Rosado, and H. Wardmann. 2004. Peripheral development of B cells in mouse and man. Immunol. Rev. 197:179-191.[CrossRef][Medline]
7. Gauld, S. B., J. M. Dal Porto, and J. C. Cambier. 2002. B-cell antigen receptor signaling: roles in cell development and disease. Science 296:1641-1642.
8. Geyer, R., S. Wee, S. Anderson, J. Yates, and D. A. Wolf. 2003. BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Mol. Cell 12:783-790.[CrossRef][Medline]
9. Gupta-Rossi, N., S. Storck, P. J. Griebel, C. A. Reynaud, J. C. Weill, and A. Dahan. 2003. Specific over-expression of deltex and a new Kelch-like protein in human germinal center B cells. Mol. Immunol. 39:791-799.[CrossRef][Medline]
10. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, and 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.
11. Hardy, R. R., and S. A. Shinton. 2004. Characterization of B lymphopoiesis in mouse bone marrow and spleen. Methods Mol. Biol. 271:1-24.[Medline]
12. Itoh, K., N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J. D. Engel, and M. Yamamoto. 1999. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13:76-86.
13. Kelso, R. J., A. M. Hudson, and L. Cooley. 2002. Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation. J. Cell Biol. 156:703-713.
14. Kraus, M., M. B. Alimzhanov, N. Rajewsky, and K. Rajewsky. 2004. Survival of resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer. Cell 117:787-800.[CrossRef][Medline]
15. Kurosaki, T. 1999. Genetic analysis of B-cell antigen receptor signaling. Annu. Rev. Immunol. 17:555-592.[CrossRef][Medline]
16. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, and R. Carsetti. 1999. B-cell development in the spleen takes place in discrete steps and is determined by the quality of B-cell receptor-derived signals. J. Exp. Med. 190:75-89.
17. McNamee, H. P., D. E. Ingber, and M. A. Schwartz. 1993. Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown. J. Cell Biol. 121:673-678.
18. Motoike, T., S. Loughna, E. Perens, B. L. Roman, W. Liao, T. C. Chau, C. D. Richardson, T. Kawate, J. Kuno, B. M. Weinstein, D. Y. Stainier, and T. N. Sato. 2000. Universal GFP reporter for the study of vascular development. Genesis 28:75-81.[CrossRef][Medline]
19. Pintard, L., J. H. Willis, A. Willems, J. L. Johnson, M. Srayko, T. Kurz, S. Glaser, P. E. Mains, M. Tyers, B. Bowerman, and M. Peter. 2003. The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425:311-316.[CrossRef][Medline]
20. Reth, M., and J. Wienands. 1997. Initiation and processing of signals from the B-cell antigen receptor. Annu. Rev. Immunol. 15:453-479.[CrossRef][Medline]
21. Rickert, R. C., J. Roes, and K. Rajewsky. 1997. B-lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25:1317-1318.
22. Rodriguez, C. I., F. Buchholz, J. Galloway, R. Sequerra, J. Kasper, R. Ayala, A. F. Stewart, and S. M. Dymecki. 2000. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25:139-140.[CrossRef][Medline]
23. Sakai, K., and J. Miyazaki. 1997. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem. Biophys. Res. Commun. 237:318-324.[CrossRef][Medline]
24. Sato, T. N., and S. Bartunkova. 2000. Analysis of embryonic vascular morphogenesis. Methods Mol. Biol. 137:223-233.[Medline]
25. Sohn, H. W., H. Gu, and S. K. Pierce. 2003. Cbl-b negatively regulates B-cell antigen receptor signaling in mature B cells through ubiquitination of the tyrosine kinase Syk. J. Exp. Med. 197:1511-1524.
26. Stogios, P. J., and G. G. Prive. 2004. The BACK domain in BTB-kelch proteins. Trends Biochem. Sci. 29:634-637.[CrossRef][Medline]
27. Storck, S., F. Delbos, N. Stadler, C. Thirion-Delalande, F. Bernex, C. Verthuy, P. Ferrier, J. C. Weill, and C. A. Reynaud. 2005. Normal immune system development in mice lacking the Deltex-1 RING finger domain. Mol. Cell. Biol. 25:1437-1445.
28. Swiatek, P. J., and T. Gridley. 1993. Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 7:2071-2084.
29. Timmerman, V., P. De Jonghe, and C. Van Broeckhoven. 2000. Of giant axons and curly hair. Nat. Genet. 26:254-255.[CrossRef][Medline]
30. Wakabayashi, N., K. Itoh, J. Wakabayashi, H. Motohashi, S. Noda, S. Takahashi, S. Imakado, T. Kotsuji, F. Otsuka, D. R. Roop, T. Harada, J. D. Engel, and M. Yamamoto. 2003. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat. Genet. 35:238-245.[CrossRef][Medline]
31. Xu, L., Y. Wei, J. Reboul, P. Vaglio, T. H. Shin, M. Vidal, S. J. Elledge, and J. W. Harper. 2003. BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425:316-321.[CrossRef][Medline]
32. Xue, F., and L. Cooley. 1993. kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell 72:681-693.[CrossRef][Medline]
33. Yan, W., L. Ma, K. H. Burns, and M. M. Matzuk. 2004. Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice. Proc. Natl. Acad. Sci. USA 101:7793-7798.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
