Gene Targeting RhoA Reveals Its Essential Role in Coordinating Mitochondrial Function and Thymocyte Development

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 due to 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 NAC partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis.

Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

“…The floxed allele contains loxP sites flanking exon 3 of the RhoA allele. To delete RhoA in vivo in T cells, RhoA flox/flox mice were mated with mice expressing Cre recombinase under the control of a CD2 proximal promoter (Jackson Laboratory, Bar Harbor, ME) . In each experiment, age‐ (8‐ to 10‐week old) and sex‐matched RhoA flox/flox CD2 Cre+ mice (hereafter, referred to as RhoA −/− or RhoA‐deficient mice) and their WT littermates (RhoA flox/flox CD2 Cre− , referred to as WT mice) were used.…”

“…Recently, we have generated a conditional RhoA‐deficient mouse line in which RhoA is specifically deleted in T cells. With this mouse model, we found that RhoA was required for coordinating mitochondrial function and thymocyte development . Moreover, RhoA orchestrated glycolysis for Th2 cell differentiation and allergic airway inflammation .…”

T‐cell activation RhoGTPase‐activating protein plays an important role in TH17‐cell differentiation

“…Rho family GTPases shift between the GTP‐bound ‘on’ and GDP‐bound ‘off’ states, thereby functioning as molecular switches for signal transduction in numerous cellular processes. Rho family GTPases, such as Rac1/2, 1 , 2 RhoA, 3 RhoH, 4 and Cdc42, 5 are involved in T‐cell receptor (TCR)‐mediated signal transduction; therefore, they play an important role in T‐cell development and activation. The intrinsic GTP hydrolysis activity of classical RhoGTPases is enhanced by RhoGTPase‐activating proteins (RhoGAPs).…”

Activation of cyclin D1 affects mitochondrial mass following traumatic brain injury

“…mtDNA will be isolated by ethanol precipitation. An aliquot of homogenates was reserved for protein quantification, and mtDNA content was normalized to the protein concentration (Cheng et al, 2012; Zhang et al, 2014). Total nuclear DNA was isolated from the nuclear fraction using QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s protocol.…”