Gene Targeting of RhoA Reveals Its Essential Role In Lymphopoiesis

Abstract 282

RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR and BCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in lymphocyte development remains unclear. In this study, we have achieved T cell, B cell, and hematopoietic stem cell-specific deletion of RhoA by conditional gene targeting with CD2, CD19 and Mx1 promoter-driven Cre expression, respectively, in the RhoA^loxP/loxP mice. First, we found that RhoA gene disruption in early T cells caused a drastic decrease in thymocyte cellularity, with the numbers of CD4⁻CD8⁻ double negative (DN), CD4⁺CD8⁺ double positive (DP), CD4⁺CD8⁻ single positive (SP), and CD4⁻CD8⁺ SP T cells decreased by 88.8% ± 6.0%, 99.4% ± 1.0%, 99.3% ± 1.2%, and 98.6% ± 2.0%, respectively. Among DN subpopulations, CD44⁺CD25⁻ (DN1), CD44⁺CD25⁺ (DN2), CD44⁻CD25⁺ (DN3), and CD44⁻CD25⁻ (DN4) cells were reduced by 91.7% ± 6.0%, 54.9% ± 27.7%, 50.9% ± 33.3%, and 96.7% ± 3.4%, respectively. Further, RhoA knockout led to a significant loss of DP thymocytes at the initial stage (CD69^highTCR^int) of positive selection, suggesting that RhoA is required for positive selection. The decreased thymocyte cellularity in mutant mice is associated with increased apoptosis of all thymic T lineages. RhoA deficiency also resulted in a perturbation in thymocyte cell cycle progression as manifested by increased BrdU incorporation in DN1 and DN2 cells and decreased BrdU incorporation in DN4 and DP cells. Concomitantly, RhoA-deficient thymocytes showed a 59.8% ± 26.3% reduction in proliferative potential in response to TCR crosslinking. Western blot analysis revealed that the activities of ZAP70, LAT, Akt, Erk, and p38 were impaired in RhoA^-/- thymocytes. In periphery, spleens of the RhoA null mice contained 7.4% ± 8.0% of CD4⁺ T cells and 3.7% ± 2.7% of CD8⁺ T cells compared with that of wild type (WT) mice. Loss of peripheral mature T cells in mutant mice is reflected by a marked reduction of naive T cells, whereas effector and memory phenotype cells were marginally affected by RhoA deficiency. RhoA-deficient naïve T cells were more susceptible to apoptosis, suggesting that homeostatic defect of naïve T cells in RhoA^-/- mice is attributed to impaired cell survival. Abrogation of RhoA caused an increased in vivo BrdU incorporation in naïve T cell compartments. Thus, RhoA deficiency induces naïve T cell homeostatic proliferation, possibly due to a compensatory effect of lymphopenia. In contrast to that in thymocytes, Erk was constitutively activated in RhoA-deficient splenic T cells. These observations implicate RhoA in the multiple stages of T cell development and the proper assembly of early TCR signaling complex. Second, deletion of RhoA in pre-proB cells had no effect on early B cell development in bone marrow but significantly inhibited late B cell development in spleen, resulting in 78.2% ± 13.6%, 78.6% ± 16.9%, and 93.2% ± 3.4% reduction in transitional, follicular, and marginal zone B cells, respectively. Plasma cells in spleen were decreased by 50.9 % ± 25.9% in RhoA null mice. However, we did not detect any changes in survival of in vivo RhoA^-/- B cells or RhoA^-/- B cells cultured in vitro with survival factor BAFF. Distinct from previously characterized Cdc42 knockout mice, BAFF-R expression was not altered in RhoA^-/- B cells. Moreover, RhoA^-/- B cells appeared to be normal in proliferation and Akt and Erk activation in response to BCR crosslinking. These data suggest that RhoA is important for late B cell development through regulation of differentiation but not cell survival or proliferation. Finally, deletion of RhoA from hematopoietic stem cells did not affect common lymphoid progenitor production, indicating that RhoA is not required for early lymphoid progenitor commitment. Taken together, these lineage-specific mouse genetic studies demonstrate that RhoA critically regulates T and B cell development by distinct cellular mechanisms at multiple stages of lymphopoiesis.