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Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

Tamehiro N, Oda H, Shirai M, Suzuki H - PLoS ONE (2015)

Bottom Line: Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells.Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells.This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, Chiba, Japan.

ABSTRACT
RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

No MeSH data available.


Related in: MedlinePlus

The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH-/- mice.(A) Analysis of RhoH protein expression by western blot in RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes. (B, C) Analysis of RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4-CD8- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4+CD8+ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001
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pone.0131047.g001: The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH-/- mice.(A) Analysis of RhoH protein expression by western blot in RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes. (B, C) Analysis of RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4-CD8- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4+CD8+ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001

Mentions: We established hCD2 promoter-driven RhoHtg mice that expressed an N-terminal HA-tagged RhoH protein. To confirm whether HA-tagged RhoH protein could function normally, RhoHtg mice were bred to RhoH-/- mice. In the RhoH-/-RhoHtg mice, protein expression of RhoH in total thymocytes was increased two-fold compared to wild-type mice (Fig 1A). Like wild-type non-tagged RhoH protein, most of the HA-tagged RhoH protein in RhoHtg thymocytes localized to the plasma membrane (S1A Fig). As has been published before, RhoH deficient mice showed severe inhibition of T cell development (Fig 1B and 1C) with thymic cellularity and the percentage of CD4SP and CD8SP cells being significantly reduced [4, 5]. We found that introduction of an HA-tagged RhoH transgene into RhoH-/- mice successfully restored cellularity and the differentiation of CD4SP and CD8SP cells (Fig 1B and 1C). The expression of the transgene were able to restore both defective positive and β-selection (Fig 1B). Expression of CD2 and CD5 on DP thymocytes, which was severely reduced in RhoH-/- [4, 5], was restored by the transgene as well (Fig 1D). The HA-tagged RhoH associated with phosphorylated Lck without stimulation, and was phosphorylated and bound to ZAP70 upon TCR-stimulation (S1B Fig), exactly the same as non-tagged endogenous RhoH. Furthermore, impaired TCR-induced phosphorylation of ERK in RhoH-/- thymocytes was restored by the HA-tagged RhoH transgene (S1C Fig). Collectively, these results demonstrate that HA-tagged RhoH is functionally equivalent to the endogenous one, indicating that HA-RhoH transgenic mice are a proper model for analyzing the molecular functions of RhoH.


Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint.

Tamehiro N, Oda H, Shirai M, Suzuki H - PLoS ONE (2015)

The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH-/- mice.(A) Analysis of RhoH protein expression by western blot in RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes. (B, C) Analysis of RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4-CD8- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4+CD8+ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001
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pone.0131047.g001: The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH-/- mice.(A) Analysis of RhoH protein expression by western blot in RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes. (B, C) Analysis of RhoH-/-, RhoH-/-RhoHTg, and RhoH+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4-CD8- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4+CD8+ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001
Mentions: We established hCD2 promoter-driven RhoHtg mice that expressed an N-terminal HA-tagged RhoH protein. To confirm whether HA-tagged RhoH protein could function normally, RhoHtg mice were bred to RhoH-/- mice. In the RhoH-/-RhoHtg mice, protein expression of RhoH in total thymocytes was increased two-fold compared to wild-type mice (Fig 1A). Like wild-type non-tagged RhoH protein, most of the HA-tagged RhoH protein in RhoHtg thymocytes localized to the plasma membrane (S1A Fig). As has been published before, RhoH deficient mice showed severe inhibition of T cell development (Fig 1B and 1C) with thymic cellularity and the percentage of CD4SP and CD8SP cells being significantly reduced [4, 5]. We found that introduction of an HA-tagged RhoH transgene into RhoH-/- mice successfully restored cellularity and the differentiation of CD4SP and CD8SP cells (Fig 1B and 1C). The expression of the transgene were able to restore both defective positive and β-selection (Fig 1B). Expression of CD2 and CD5 on DP thymocytes, which was severely reduced in RhoH-/- [4, 5], was restored by the transgene as well (Fig 1D). The HA-tagged RhoH associated with phosphorylated Lck without stimulation, and was phosphorylated and bound to ZAP70 upon TCR-stimulation (S1B Fig), exactly the same as non-tagged endogenous RhoH. Furthermore, impaired TCR-induced phosphorylation of ERK in RhoH-/- thymocytes was restored by the HA-tagged RhoH transgene (S1C Fig). Collectively, these results demonstrate that HA-tagged RhoH is functionally equivalent to the endogenous one, indicating that HA-RhoH transgenic mice are a proper model for analyzing the molecular functions of RhoH.

Bottom Line: Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells.Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells.This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, Chiba, Japan.

ABSTRACT
RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

No MeSH data available.


Related in: MedlinePlus