| Abstract |
The aim of the present work was studying the transcriptional regulation mechanisms of the small RhoA and RhoB Rho GTPases as well as the possible role of the TGFβ signaling pathway in regulating the expression of these genes in stem cells.
Small GTPases of the Rho subfamily have been implicated in many physiological and pathological processes in various cell types including embryonic stem cells, and their deregulation may lead to diseases such as cancer. In the present study we have performed a functional analysis of the promoters of the RhoA and the RhoB genes in order to identify regulatory elements that are important for their transcriptional control in normal (CGR8) and cancer (P-19) embryonic stem cells. We first showed that the mRNA levels of RhoA were significantly higher compared with the mRNA levels of RhoB in both embryonic stem cell lines as well in various cancer cell lines and this difference could be accounted for, at least in part, by differences in the activities of the corresponding promoters. A deletion analysis of the RhoA and RhoB promoters in CGR8 and P-19 cells revealed that the proximal regions of both promoters appear to contain regulatory elements that are critical for their activity. Notably, both proximal promoters contain CCAAT boxes and mutagenesis of these elements decreased significantly the activity of both promoters suggesting a coordinated regulation of the two genes by CCAAT box binding factors. Finally, we showed that both genes are subject to transcriptional autoregulation in stem cells and in the case of RhoB, this autoregulation requires the GTPase activity of the Rho proteins.
TGFβ is a member of the transforming growth factor family, including Inhibins, Activin, AMH, BMPs, GDFs and Nodal. Generally, TGFs act as morphogens during embryogenesis providing positional information for axons determination. First of all, we examined the possible TGFβ induction of the RhoB gene in normal mouse stem cells which was not feasible under standard culture conditions, in the presence of LIF factor which preserve stem cell pluripotency. Concomitantly, under those conditions stem cells were not possible to differentiate by TGFβ. On the contrary, reduction of LIF allowed transcriptional induction of the RhoB gene by TGFβ, implying that LIF and TGFβ pathways act in opposing ways. Subsequently, we tried to repress TGFβ pathway
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in complete absence of LIF factor in order to investigate the role of TGFβ in the differentiation process. We observed that inhibition of the TGFβ pathway allowed pluripotency maintenance of the stem cells despite LIF absence, opposite to control cells that were committed to mesodermal differentiation. However, under those conditions there was no alteration in the transcriptional regulation of Rho genes.
In summary, despite the difference in the levels of expression of the RhoA and RhoB genes in mouse embryonic stem cells, their promoters are regulated by common mechanisms. However, certain aspects of RhoA and RhoB regulation, especially their autoregulation, differ among normal and cancer stem cells. Furthermore, TGFβ induction of the RhoB gene was possible only in differentiation culture conditions. Although TGFβ inhibition allowed stem cell pluripotency maintenance, RhoA and RhoB gene expression was not altered. Understanding the mechanisms that control the transcription of Rho GTPases in stem cells is necessary in order to clarify their role in stem cell functions as well as in cancer cells that share many features with stem cells.
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