PMID:25398325

Citation	Zhang, S, Konstantinidis, DG, Yang, JQ, Mizukawa, B, Kalim, K, Lang, RA, Kalfa, TA, Zheng, Y and Guo, F (2014) Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development. J. Immunol. 193:5973-82
Abstract	Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival. Nonetheless, the physiological function of RhoA in thymocyte development is not clear. By characterizing a conditional gene targeting mouse model bearing T cell deletion of RhoA, we show that RhoA critically regulates thymocyte development by coordinating multiple developmental events. RhoA gene disruption caused a strong developmental block at the pre-TCR checkpoint and during positive selection. Ablation of RhoA led to reduced DNA synthesis in CD4(-)CD8(-), CD4(+)CD8(-), and CD4(-)CD8(+) thymocytes but not in CD4(+)CD8(+) thymocytes. Instead, RhoA-deficient CD4(+)CD8(+) thymocytes showed an impaired mitosis. Furthermore, we found that abrogation of RhoA led to an increased apoptosis in all thymocyte subpopulations. Importantly, we show that the increased apoptosis was resulted from reduced pre-TCR expression and increased production of reactive oxygen species (ROS), which may be because of an enhanced mitochondrial function, as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, and mitochondrial biogenesis in RhoA-deficient thymocytes. Restoration of pre-TCR expression or treatment of RhoA-deficient mice with a ROS scavenger N-acetylcysteine partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate, to our knowledge, for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis.
Links	PubMed PMC4258484 Online version:10.4049/jimmunol.1400839
Keywords	Animals; Antigens, Surface; Apoptosis/genetics; Apoptosis/immunology; Cell Differentiation; Cell Lineage/genetics; Cell Lineage/immunology; Cell Survival/genetics; Gene Expression Profiling; Gene Targeting; Immunophenotyping; Mice; Mice, Knockout; Mitochondria/genetics; Mitochondria/metabolism; Phenotype; Receptors, Antigen, T-Cell, alpha-beta/genetics; Receptors, Antigen, T-Cell, alpha-beta/metabolism; T-Lymphocyte Subsets/cytology; T-Lymphocyte Subsets/metabolism; Thymocytes/cytology; Thymocytes/metabolism; V(D)J Recombination; rhoA GTP-Binding Protein/deficiency; rhoA GTP-Binding Protein/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MOUSE:RHOA		GO:0046638: positive regulation of alpha-beta T cell differentiation	ECO:0000315:		P		Fig 2B: the DN4:DN3 ratio decreased in mice without RhoA, indicating that transition from DN3 to DN4 (beta selection) is blocked RhoA absence Fig 3B: apoptosis of DN4 cells increased to a larger extent than apoptosis of DN3 cells in mice without RhoA	complete CACAO 10938
MOUSE:RHOA		GO:0043366: beta selection	ECO:0000315:		P		Fig 2B: the DN4:DN3 ratio decreased in mice without RhoA, indicating that transition from DN3 to DN4 (beta selection) is blocked RhoA absence Fig 3B: apoptosis of DN4 cells increased to a larger extent than apoptosis of DN3 cells in mice without RhoA	complete CACAO 10939
MOUSE:RHOA		GO:0032956: regulation of actin cytoskeleton organization	ECO:0000315:		P		Fig 5D: little/no F-actin was found in multinucleus cells without RhoA	complete CACAO 10942
MOUSE:RHOA		GO:0070507: regulation of microtubule cytoskeleton organization	ECO:0000315:		P		Fig 5E: beta-tubulin was not found in multinucleus cells without RhoA Fig 5F: fluoresence of beta-tubulin decreased in single nucleus cells without RhoA	complete CACAO 10943
MOUSE:RHOA		GO:1902234: positive regulation of positive thymic T cell selection	ECO:0000315:		P		Fig 4B: more TCRint CD69+ (double positive cells at the beginning of positive selection) than TCRhi CD69+ and TCRhi CD69- (immature and mature single positive cells after positive selection) in cells without RhoA - the ratio of these cells in the wildtype is closer to 1:1:1 Fig 4C: p14TCR mice had fewer CD8+ SP cells than wildtype - p14TCR cells have a mutation in TCRVa2Vb8 that causes them to undergo positive selection at a higher rate than wildtype cells, so they normally form a lot of CD8+ SP cells (result of positive selection and the subsequent transition phase)	complete CACAO 10944
MOUSE:RHOA		GO:0002363: alpha-beta T cell lineage commitment	ECO:0000315:		P		Fig 4D: fewer CD4+ CD8int cells are formed from cells without RhoA - RhoA affects lineage commitment in cells lineage commitment involves the transformation of double positive (DP) cells to CD4+ CD8int and the subsequent transformation of CD4+ CD8int to either CD4+ SP or CD8+ SP	complete CACAO 10945
MOUSE:RHOA		GO:0090324: negative regulation of oxidative phosphorylation	ECO:0000315:		P		Fig 6A: OCR (associated with oxidative phosphorylation) levels increased in cells without RhoA Fig 6B: amount of mRNA for various genes associated with oxidative phosphorylation increased in cells without RhoA	complete CACAO 10947
MOUSE:RHOA		GO:0045820: negative regulation of glycolytic process	ECO:0000315:		P		Fig 5C: level of ECAR (associated with glycolysis) increased in cells without RHoA Fig 5D: more mRNA for genes associated with glycolysis was produced in cells without RhoA	complete CACAO 10948
MOUSE:RHOA		GO:1903427: negative regulation of reactive oxygen species biosynthetic process	ECO:0000315:		P		Fig 7A: reactive oxygen species (ROS) levels increased in cells without RhoA	complete CACAO 10949
MOUSE:RHOA	involved_in	GO:0046638: positive regulation of alpha-beta T cell differentiation	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:0043366: beta selection	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:0032956: regulation of actin cytoskeleton organization	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:0070507: regulation of microtubule cytoskeleton organization	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:0002363: alpha-beta T cell lineage commitment	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:0090324: negative regulation of oxidative phosphorylation	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:RHOA	involved_in	GO:1903427: negative regulation of reactive oxygen species biosynthetic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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