Previous work has shown that RhoH is a negative regulator for growth, survival and cytoskeletal modifications . We show here that the expression level of RhoH modulates the activity of STAT transcription factors STAT5 and STAT1. In the IL3-dependent cell line BaF3, RhoH acts as a specific negative regulator of IL3, but not Epo-induced proliferation and silencing of RhoH gene expression allows the cells to proliferate faster in response to IL3.
The JAK-STAT pathway is a major signalling pathway of haematopoietic cells that links proliferative signals to the cell cycle machinery. In IL3-mediated signalling, STAT5 plays a major role in the regulation of proliferation, differentiation and anti-apoptotic signalling [25, 26]. We demonstrate that overexpression of RhoH leads to a decrease in the activity of STAT5, whereas silencing of RhoH expression causes an increased activity of STAT5 compared to control cells. No changes in the expression level of total STAT5 protein were detectable and we therefore conclude that RhoH does not modulate STAT5 activity through regulation of STAT5 expression levels. Most interestingly, we also could show a link between RhoH expression levels and changes in the surface expression of the ILR3 α-chain CD123.
It had previously been suggested that an elevated CD123 expression, as it can be found in patients with acute myeloid leukaemia (AML), may contribute to the increased proliferation of leukaemic blasts, hyperactivation of STAT5 and poor prognosis . Low expression levels of RhoH were recently described as yet another factor linked to poor patient prognosis . Our data now show that these two findings indeed might be connected. Because low RhoH expression leads to an increased STAT5 activity, STAT5 might then induce expression of the IRF-1 gene, which in turn allows an IRF-1-dependent upregulation of the CD123 gene, eventually leading to an increase in the surface levels of the protein.
Although the regulatory influence of RhoH on STAT5 activity would be sufficient to account for the differences in proliferation, we observed an additional mechanism by which RhoH negatively regulates IL3-induced growth, namely the activation of STAT1 in RhoH overexpressing cells. STAT1 is the key factor that transduces the antiproliferative effects of interferons  and activation of STAT1 coincides with cell cycle arrest or apoptosis. As a consequence, STAT1 knock-out mice develop tumours more rapidly [32, 33]. When we screened control cells and RhoH overexpressing cells for differences in their sensitivity towards apoptotic stimuli, we were not able to find any. However, siRhoH showed increased survival after cytokine deprivation and readdition of IL3 to starved cells induced a pool of rapidly growing cells, whereas parental cells did not recover (unpublished data).
It has been reported that STAT1 activation can lead to the upregulation of p21
causing subsequent cell cycle arrest or apoptosis and a STAT1 DNA binding site was found in the p21
promoter . Another member of this family, p27
, was shown to be downregulated by IL3 and BCR-ABL . Interestingly, we found that p21
are both upregulated when RhoH is expressed, i.e. STAT1 is activated, and we suggest this as a RhoH-dependent mechanism that serves to regulate progression in the cell cycle. We propose a model, where the balance between proliferation and apoptosis is fine-tuned by the expression level of RhoH. While high levels of RhoH lead to increased STAT1 but reduced STAT5 activity, downregulation of RhoH expression activates STAT5-dependent proliferation and survival signals. It will be important to examine whether in IL3 sensitive, differentiating haematopoietic progenitor cells, the expression level of RhoH can regulate the balance between proliferation and cell cycle arrest or apoptosis. There was no obvious haematopoietic defect in RhoH-deficient animals, however, it is possible that the disturbed IL3-dependent signalling can be compensated by other cytokines. In addition, it is known that in B cells, RhoH is a target of somatic hypermutation and translocation which affects the expression of the protein . Nevertheless, RhoH-deficient animals did not develop lymphomas or show other B cell malignancies, which is a discrepancy that shows the limit of the animal model.
Two recent publications now link low RhoH protein levels to cancer [12, 13]. In AML, RhoH expression is low, causing high levels of active, GTP-bound Rac1 and eventually resistance to chemotherapeutic apoptosis . Our results indicate that other signalling pathways, such as STAT5 activation and high expression of the IL3-binding α chain, might additionally be modulated by RhoH and contribute to the disease. To understand the importance of RhoH for the development of haematopoietic malignancies, it will be crucial to establish a link between RhoH mutations, its expression on the protein level and the activity of signalling molecules such as STATs that are known to be upregulated in a number of myeloproliferative disorders [35, 36]. In addition, the JAK-STAT pathway plays a central role in cytokine-mediated signalling in haematopoiesis and the immune system. This pathway has not yet been discussed as a potential target of RhoH and it will therefore be interesting to see whether cytokine receptors other than IL3 are regulated through the expression level of RhoH.