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RhoA of the Rho family small GTPases is essential for B lymphocyte development.

Zhang S, Zhou X, Lang RA, Guo F - PLoS ONE (2012)

Bottom Line: Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion.Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation.Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, Cincinnati, Ohio, United States of America.

ABSTRACT
RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.

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RhoA is necessary for BAFF-mediated B cell survival but not proliferation.(A) Splenic B220+ B cells from CD19Cre/+; RhoA+/+ (control) and CD19Cre/+; RhoAflox/flox (RhoA−/−) mice were cultured for 48 hours on 96-well plates (4×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD490. n = 5. (B) Splenocytes from control and RhoA−/− mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220+ B cells from control and RhoA−/− mice were cultured for 72 hours on 96-well plates (2×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and RhoA−/− mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR+ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR+ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220+ B cells from control and RhoA−/− mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220+ B cells from control and RhoA−/− mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220+ B cells from control and RhoA−/− mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.
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pone-0033773-g002: RhoA is necessary for BAFF-mediated B cell survival but not proliferation.(A) Splenic B220+ B cells from CD19Cre/+; RhoA+/+ (control) and CD19Cre/+; RhoAflox/flox (RhoA−/−) mice were cultured for 48 hours on 96-well plates (4×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD490. n = 5. (B) Splenocytes from control and RhoA−/− mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220+ B cells from control and RhoA−/− mice were cultured for 72 hours on 96-well plates (2×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and RhoA−/− mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR+ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR+ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220+ B cells from control and RhoA−/− mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220+ B cells from control and RhoA−/− mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220+ B cells from control and RhoA−/− mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.

Mentions: Cell proliferation is crucial for B cell development [4]. To explore the mechanisms underlying impaired splenic B cell development in RhoA-deficient mice, we determined the proliferative capacity of RhoA−/− splenic B cells upon in vitro culture with either LPS or anti-IgM F(ab')2 antibody to crosslink BCR. Surprisingly, we found that under either condition the RhoA−/− B cell proliferation profile did not statistically differ from control cells (Fig. 2A). These results suggest that RhoA is dispensable for Toll-like receptor- or BCR-mediated B cell proliferation. Along with recent studies showing that RhoA-deficient neural progenitor cells are hyperproliferative while RhoA-deficient MEFs have impaired proliferation [22], [23], it appears that RhoA plays cell type-specific roles in the regulation of cell proliferation.


RhoA of the Rho family small GTPases is essential for B lymphocyte development.

Zhang S, Zhou X, Lang RA, Guo F - PLoS ONE (2012)

RhoA is necessary for BAFF-mediated B cell survival but not proliferation.(A) Splenic B220+ B cells from CD19Cre/+; RhoA+/+ (control) and CD19Cre/+; RhoAflox/flox (RhoA−/−) mice were cultured for 48 hours on 96-well plates (4×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD490. n = 5. (B) Splenocytes from control and RhoA−/− mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220+ B cells from control and RhoA−/− mice were cultured for 72 hours on 96-well plates (2×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and RhoA−/− mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR+ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR+ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220+ B cells from control and RhoA−/− mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220+ B cells from control and RhoA−/− mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220+ B cells from control and RhoA−/− mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.
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pone-0033773-g002: RhoA is necessary for BAFF-mediated B cell survival but not proliferation.(A) Splenic B220+ B cells from CD19Cre/+; RhoA+/+ (control) and CD19Cre/+; RhoAflox/flox (RhoA−/−) mice were cultured for 48 hours on 96-well plates (4×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD490. n = 5. (B) Splenocytes from control and RhoA−/− mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220+ B cells from control and RhoA−/− mice were cultured for 72 hours on 96-well plates (2×105 cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')2 antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and RhoA−/− mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR+ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR+ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220+ B cells from control and RhoA−/− mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220+ B cells from control and RhoA−/− mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220+ B cells from control and RhoA−/− mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.
Mentions: Cell proliferation is crucial for B cell development [4]. To explore the mechanisms underlying impaired splenic B cell development in RhoA-deficient mice, we determined the proliferative capacity of RhoA−/− splenic B cells upon in vitro culture with either LPS or anti-IgM F(ab')2 antibody to crosslink BCR. Surprisingly, we found that under either condition the RhoA−/− B cell proliferation profile did not statistically differ from control cells (Fig. 2A). These results suggest that RhoA is dispensable for Toll-like receptor- or BCR-mediated B cell proliferation. Along with recent studies showing that RhoA-deficient neural progenitor cells are hyperproliferative while RhoA-deficient MEFs have impaired proliferation [22], [23], it appears that RhoA plays cell type-specific roles in the regulation of cell proliferation.

Bottom Line: Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion.Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation.Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, Cincinnati, Ohio, United States of America.

ABSTRACT
RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.

Show MeSH
Related in: MedlinePlus