The most obvious phenotype of the mice with RhoH gene ablation was seen in the pronounced reduction in thymocyte numbers. Since RhoH was shown to be expressed at the highest levels in the thymus, we suspected that RhoH might play a role in T cell differen- tiation or function.
Indeed, in the absence of RhoH, αβ thymocyte development was partially blocked at the DN3 to DN4 stage and at the DN4 to DP transition, leading to an accumulation of DN3 thymocytes (s. 4.4.4.1). At this transition β-selection takes place, which ensures that only thymocytes that have generated a functional TCRβ chain can differentiate to DN4 and DP cells. A second partial block occurred at the transition from DP to CD4SP and CD8SP cells, where positive selection allows only MHC-restricted, self-tolerant thymocytes to develop further. Since both β-selection and positive selection are dependent on similar signaling cascades downstream of pre-TCR and TCR, our results indicated a possible role of RhoH in these signaling pathways. Aberrant expression of maturation markers, such as CD5, TCRβ and CD69 (s. 4.4.4.6)
suggested a defective pre-TCR and TCR signaling. Furthermore, analysis of TCR-stimulated RhoH-deficient thymocytes confirmed that TCR signaling was impaired in the absence of RhoH (s. 4.6).
The phenotype of the RhoH-deficient mice was surprising, as it differed strongly from what we anticipated to find based on previously published observations. As demonstrated in two studies, RhoH suppresses Rac1 activation and also Rac1-, Cdc42- and RhoA-mediated activation of NF-κB and p38 (Gu et al., 2005a; Li et al., 2002). We rather expected a disturbance of thymocyte development associated with a constitutive activation of Rac1, NF- κB or p38. However, constitutive activation of these proteins results in thymic differentiation disorders that are different from the defects observed in the thymocyte development in the absence of RhoH, suggesting that in thymocytes in vivo, RhoH is not crucial for regulation of these signaling molecules.
Constitutive activation of Rac1 increases proliferation of DN3 thymocytes and accelerates DN3 to DN4 transition by potentiation of pre-TCR signaling (Gomez et al., 2000). At later stages, constitutively active (c.a.) Rac1 enhances TCR signaling and thereby diverts positive selection to negative selection leading to decreased numbers of SP cells. Due to augmented TCR signaling, DP and SP thymocytes have a higher percentage of cells with upregulated expression of CD5 and CD69. In contrast, RhoH-deficient thymocytes revealed attenuated pre-TCR and TCR signaling (s. 4.6), resulting in a partial block from DN3 to DN4 and from DN4 to DP stages (s. 4.4.4.1) as well as in an impaired positive selection (s. 4.5.2). Although we observed an increased basal activation of Rac1 in the absence of RhoH (s. 4.6.4), the different phenotypes of c.a. Rac1 transgenic and RhoH-null mice suggest that the altered Rac1 activation is not contributing to the defective thymocyte development.
If RhoH-deficiency would cause constitutive activation of NF-κB in thymocytes, we would expect an increase of DN4 cells (Voll et al., 2000). NF-κB is activated through several receptors leading in a classical pathway to activation of the IκB kinase complex (IKKα, IKKβ, IKKγ), which phosphorylates IκB-α, the NF-κB inhibitor. Phosphorylated IκB-α is then ubiquitinylated and degraded by the proteasome, thus releasing NF-κB for translocation to the nucleus and activation of target genes (Siebenlist et al., 2005). Enhanced activation of NF-κB, as achieved by the expression of a c.a. IKKβ, provides a survival signal for the DN3 cells and leads to an increase in the DN4 population (Voll et al., 2000). In fact, we observed a decreased survival of DN4 thymocytes in RhoH-deficient mice.
The effect of augmented NF-κB activity on negative and positive selection was not examined in mice expressing c.a. IKKβ, but studies involving overexpression of the inhibitor IκB-α or
the d.n. form of IKK propose that NF-κB might be involved in both negative and positive selection by promoting apoptosis of autoreactive thymocytes and survival of positively selected cells (Siebenlist et al., 2005). All these effects were not observed in RhoH-null thymocytes.
Finally, if the consequence of ablation of the RhoH gene was elevated p38 MAP kinase activity, RhoH-deficient mice should show a progressive increase of thymus size, causing extinction of lung expansion and death of the mice around 5-6 months of age, as was shown for mice constitutively expressing MAP kinase kinase 6 (MKK6) in thymocytes, which specifically activates p38 (Diehl et al., 2000; Rincon and Pedraza-Alva, 2003).
Inactivation of p38 is required for the DN3 to DN4 transition. Persistent activation of p38 leads to a continuous accumulation of immature DN3 cells and an arrest in further develop- ment to DN4 and DP cells during fetal thymic differentiation, resulting in a complete absence of mature T cells in the periphery (Diehl et al., 2000; Rincon and Pedraza-Alva, 2003). Intro- duction of MKK6 into developing thymocytes forces deletion of DP cells, suggesting that p38 promotes negative selection. (Sugawara et al., 1998). Although we detected a significant increase of DN3 thymocytes in the absence of RhoH which could coincide with enhanced p38 activity, no complete arrest of thymocyte development occurred. Most importantly, we could not observe increased activation of p38 in RhoH-null DP cells (s. 4.6.5), making it unlikely that p38 activity is elevated in RhoH-deficient DN3 cells.
Taken together, the defects detected in the thymocyte development in RhoH-null mice did not match our expectations on the effect of RhoH deletion, thus establishing a novel role of RhoH as a positive regulator of T cell development.