Abstract
The hematopoietic, atypical GTPase RhoH is reported to function as a negative regulator of progenitor cell differentiation, proliferation and survival. IL-3 is an important cytokine, signalling mainly via the JAK-STAT pathway and by this promoting hematopoiesis, progenitor cell survival and differentiation of myeloid cells, such as mast cells. This study aimed to investigate the role of RhoH and important components in the signalling network of RhoH in IL-3 mediated signal transduction. It was attempted to link published, as well as the gained, information of this study to profile hematopoietic malignancies with aberrant RhoH expression. Finally, complementary studies were performed with in vitro IL-3 derived mast cells to address the role of RhoH in IL-3 mediated cell differentiation and its function in mast cell mediated processes, such as anaphylaxis. RhoH overexpression in BaF3 cells resulted in inhibition of IL-3 induced proliferation correlating with increased STAT1 and decreased STAT5 activity. Downregulation of RhoH was followed by an increase in IL-3 proliferation associated with enhanced STAT5 phosphorylation. Altered STAT1 activation induced increased expression of the cell cycle inhibitors (CDKIs) p21waf/cip and p27kip. Enforced IL-3 induced STAT5 activation in cells with low RhoH expression led to enhanced sensitivity of cells to IL-3, as a result of an upregulation of the STAT5 dependent transcription factor IRF-1 which induced increased IL-3 alpha chain (CD123) surface expression. RhoH expression was tested in lymphoma cell lines which were characterised based on their immunophenotype, STAT and GTPase activity. RhoH was strongly underexpressed in almost every cell line, suggesting that RhoH plays an important role in the formation of hematopoietic cancer types. To gain further insight into the IL-3 dependent signalling networks of RhoH, cytokine treatment dependent proteomic experimebyents were performed with IL-3 dependent BaF3 cells. A cytokine dependent pattern of protein interactions was identified. The interaction with RhoH was verified in HEK cells for two proteins: Cofilin-1 and PTP1B. Both of these proteins were found to have effects that can be related to the previous findings of this study in IL-3 mediated signalling. Finally, experiments in mast cells were performed to identify the role of RhoH for IL-3 dependent cell differentiation and function, indicating that RhoH deficiency does not affect the IL-3 induced differentiation. However, the loss of RhoH has functional consequences on mast cell biology, such as decreased cytokine production and mast cell mediator release. These findings link RhoH expression and RhoH modulated IL-3 induced proliferation on the one hand to STAT1 dependent induction of CDKIs and on the other hand to the increased STAT5 dependent CD123 expression. It is suggested that RhoH is a tandem regulator of IL-3 dependent STAT5/STAT1 activation, most likely by newly discovered interactions with key signalling proteins such as PTP1B and Cofilin-1. Altered RhoH expression pattern in different hematopoietic disorders also suggests that the identified role of RhoH in IL-3 dependent signalling is transferrable to aberrant signalling in cancerogenous diseases. Furthermore, it was postulated that, although RhoH is dispensable for IL-3 mediated myeloid mast cell differentiation, the lack of RhoH has serious impact on mast cell mediated effects.
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