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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 required for bone marrow B cell development.(A) Mx-Cre; RhoAflox/flox(RhoA−/−) mice were injected with polyI:C to delete RhoA in HSCs (left). RhoAflox/flox mice were used as control and also subjected to polyI:C injection. Whole bone marrow was isolated from RhoA−/− and control mice and analyzed for RhoA expression by Western blot (right). (B) Bone marrow cells from RhoA−/− and control mice were stained for IL-7R, lineage (Lin) markers (B220, CD3, CD4, CD8, Gr1, CD11b and TER119), Sca-1, and c-Kit followed by flow cytometry analysis. Common lymphoid progenitor (CLP) cells (Sca1+c-kit+) were gated from a Lin−IL-7R+ cell population (left). The number of CLPs was calculated by multiplying the total number of bone marrow cells by the percentage of CLP cells (right). n = 4. (C) Bone marrow cells from RhoA−/− and control mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 4. 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-g003: RhoA is required for bone marrow B cell development.(A) Mx-Cre; RhoAflox/flox(RhoA−/−) mice were injected with polyI:C to delete RhoA in HSCs (left). RhoAflox/flox mice were used as control and also subjected to polyI:C injection. Whole bone marrow was isolated from RhoA−/− and control mice and analyzed for RhoA expression by Western blot (right). (B) Bone marrow cells from RhoA−/− and control mice were stained for IL-7R, lineage (Lin) markers (B220, CD3, CD4, CD8, Gr1, CD11b and TER119), Sca-1, and c-Kit followed by flow cytometry analysis. Common lymphoid progenitor (CLP) cells (Sca1+c-kit+) were gated from a Lin−IL-7R+ cell population (left). The number of CLPs was calculated by multiplying the total number of bone marrow cells by the percentage of CLP cells (right). n = 4. (C) Bone marrow cells from RhoA−/− and control mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 4. 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: As previously demonstrated, CD19Cre/+-mediated RhoA deletion in bone marrow B cells is inefficient (Fig. 1A), precluding further examination of the role of RhoA in their development. Instead, we employed an alternatively method to generate RhoA−/− HSCs by crossing RhoAflox/flox with Mx-Cre transgenic mice and inducing Cre expression in Mx-Cre; RhoAflox/flox mice with polyI:C (Fig. 3A). RhoAflox/flox littermates were used as control and also treated with polyI:C. Since Mx-Cre; RhoAflox/flox mice die within eight days after polyI:C induction, we analyzed the mice at day six and found that RhoA was almost completely absent from the bone marrow (Fig. 3A). FACS analysis indicated that both the percentage and number of common lymphoid progenitors (CLP) were elevated in Mx-Cre; RhoAflox/flox mice (Fig. 3B), but the proB/preB and immature B cell subsets were markedly decreased (Fig. 3C). The percentage, but not number, of recirculating mature B cells in bone marrow was increased in Mx-Cre; RhoAflox/flox mice (Fig. 3C and data not shown), likely due to a compensatory effect of the decreased proB/preB and immature B cells. These results suggest that RhoA deficiency inhibits bone marrow B cell development at the CLP differentiation stage. Thus, RhoA also plays a critical role in early B cell development.


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 required for bone marrow B cell development.(A) Mx-Cre; RhoAflox/flox(RhoA−/−) mice were injected with polyI:C to delete RhoA in HSCs (left). RhoAflox/flox mice were used as control and also subjected to polyI:C injection. Whole bone marrow was isolated from RhoA−/− and control mice and analyzed for RhoA expression by Western blot (right). (B) Bone marrow cells from RhoA−/− and control mice were stained for IL-7R, lineage (Lin) markers (B220, CD3, CD4, CD8, Gr1, CD11b and TER119), Sca-1, and c-Kit followed by flow cytometry analysis. Common lymphoid progenitor (CLP) cells (Sca1+c-kit+) were gated from a Lin−IL-7R+ cell population (left). The number of CLPs was calculated by multiplying the total number of bone marrow cells by the percentage of CLP cells (right). n = 4. (C) Bone marrow cells from RhoA−/− and control mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 4. 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-g003: RhoA is required for bone marrow B cell development.(A) Mx-Cre; RhoAflox/flox(RhoA−/−) mice were injected with polyI:C to delete RhoA in HSCs (left). RhoAflox/flox mice were used as control and also subjected to polyI:C injection. Whole bone marrow was isolated from RhoA−/− and control mice and analyzed for RhoA expression by Western blot (right). (B) Bone marrow cells from RhoA−/− and control mice were stained for IL-7R, lineage (Lin) markers (B220, CD3, CD4, CD8, Gr1, CD11b and TER119), Sca-1, and c-Kit followed by flow cytometry analysis. Common lymphoid progenitor (CLP) cells (Sca1+c-kit+) were gated from a Lin−IL-7R+ cell population (left). The number of CLPs was calculated by multiplying the total number of bone marrow cells by the percentage of CLP cells (right). n = 4. (C) Bone marrow cells from RhoA−/− and control mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 4. 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: As previously demonstrated, CD19Cre/+-mediated RhoA deletion in bone marrow B cells is inefficient (Fig. 1A), precluding further examination of the role of RhoA in their development. Instead, we employed an alternatively method to generate RhoA−/− HSCs by crossing RhoAflox/flox with Mx-Cre transgenic mice and inducing Cre expression in Mx-Cre; RhoAflox/flox mice with polyI:C (Fig. 3A). RhoAflox/flox littermates were used as control and also treated with polyI:C. Since Mx-Cre; RhoAflox/flox mice die within eight days after polyI:C induction, we analyzed the mice at day six and found that RhoA was almost completely absent from the bone marrow (Fig. 3A). FACS analysis indicated that both the percentage and number of common lymphoid progenitors (CLP) were elevated in Mx-Cre; RhoAflox/flox mice (Fig. 3B), but the proB/preB and immature B cell subsets were markedly decreased (Fig. 3C). The percentage, but not number, of recirculating mature B cells in bone marrow was increased in Mx-Cre; RhoAflox/flox mice (Fig. 3C and data not shown), likely due to a compensatory effect of the decreased proB/preB and immature B cells. These results suggest that RhoA deficiency inhibits bone marrow B cell development at the CLP differentiation stage. Thus, RhoA also plays a critical role in early B cell development.

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