Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function

Abstract

Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development is regulated by a complex signalling network [1], one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4,6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7]. In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck-C3 mice, in which the reduction predominantly results from defects in cell survival of CD25(+) thymocyte progenitors, the CD2-C3 transgenic mice had a pre-T-cell differentiation block at the CD25(+) stage after rearrangement of the T-cell receptor (TCR) beta chains. Analysis of CD2-C3 mice demonstrated that Rho acts as an intracellular switch for TCR beta selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25(+) pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.

Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes

“…In normal T cell progenitors, proliferation and differentiation are dependent on both PDK1-and RhoA-dependent signaling pathways (Cleverley et al, 1999;Hinton et al, 2004;Kelly et al, 2007;Mullin et al, 2007). The present data show that PTEN-deleted thymocytes remain RhoA dependent for proliferation, but can proliferate and differentiate without PDK1 and active PKB.…”

“…Mice were maintained in specific pathogen-free conditions under Home Office project licenses PPL60/3116 and PPL60/3812. Pten fl/fl (Marino et al, 2002), PDK1 fl/fl (Mora et al, 2003), Lck-cre (Takahama et al, 1998), Rag2 / (Shinkai et al, 1992), and transgenic CD2-C3 (Cleverley et al, 1999) mice were bred and maintained in the Wellcome Trust Biocentre/ Transgenics Resource Unit, University of Dundee in compliance with UK Home Office Animals (Scientific Procedures) Act 1986 guidelines. Pten fl/fl Lck-cre +/ and PDK1 fl/fl Lck-cre +/ mice were generated as described previously (Hagenbeek et al, 2004;Hinton et al, 2004).…”

“…In this context, it is known that PI(3,4,5)P 3 signaling can also link to signaling pathways mediated by Rac/Rho family GTPases and these are also very important regulators of thymocyte proliferation (Cleverley et al, 1999;Liliental et al, 2000;Mullin et al, 2007). The key question then is does PTEN deletion bypass all the normal signaling pathways that control T cell development?…”

“…The key question then is does PTEN deletion bypass all the normal signaling pathways that control T cell development? The importance of RhoA for thymocytes was discovered by studies of transgenic mice that express Clostridium botulinum C3-transferase under the control of T cell-specific promoters (Cleverley et al, 1999). This toxin selectively ADP-ribosylates RhoA within its effectorbinding domain and abolishes its biological function.…”

The RhoA‐ and CDC42‐specific exchange factor Dbs promotes expansion of immature thymocytes and deletion of double‐positive and single‐positive thymocytes

“…In transgenic mice expressing C3 transferase, an inhibitor of RhoA and other Rho-type GTPases but not Rac1, Rac2 or CDC42, there was a severe depletion in DN2, DN3 and DN4 thymocytes, due to increased incidence of apoptosis at the earlier stages and suppressed ability to differentiate into the DN4 stage [13][14][15][16]. The latter defect may reflect a requirement for one or more of the Rhotype GTPases in pre-TCR-mediated progression beyond the DN3 stage [14,17].…”