| Abstract |
One percent of the human genome encodes for proteins that regulate or are regulated by members of the Rho-GTPases family. Rho proteins are characterized as molecular switches that control many of the fundamental processes of cell biology like transcriptional regulation, cell growth, apoptosis and cytoskeleton reorganization during cell migration.
Whereas the classical Rho GTPases are regulated by GDP/GTP cycling, in some cases transcriptional regulation, which determines the expression levels of the proteins in the cell, plays also a crucial role in their function. RhoB is the only member of the RhoA-related subfamily that is transcriptionally regulated and this regulation seems to be of high importance for its function due to the short half-life of this protein in the cell. In a series of DNA microarray studies of TGFβ-induced cell processes like epithelial-mesenchymal transition (EMT) and apoptosis led to the discovery of RhoB as one of the most critical transcriptional targets of TGFβ pathway.
In the present study, we investigated the molecular mechanisms that control RhoB gene expression at the transcriptional level. In the first part, we sought to determine whether RhoB is transcriptionally activated by TGFβ and which TGFβ-induced pathways are responsible for this upregulation. In the second and third part, we studied the transcriptional regulation of RhoB by other transcription factors such as Sp1 and members of p53 family of tumor suppressor proteins.
The results of our study concerning the role of TGFβ in RhoB activation are concluded below:
a) TGFβ activates transcriptionally the RhoB gene in different cells types such as human keratinocytes (HaCaT), hepatoma cells (HepG2) and mouse fibroblasts (Swiss3T3) b) in this upregulation are involved both the MAP kinase pathway, specifically kinases ERK1/ERK2, and the Smad pathway but the two pathways seem to have independent roles. The pathway of MAP kinases acts during the first 3 hours of TGFβ treatment and probably it is responsible for the early eventsin RhoB transcriptional regulation. On the contrary, the Smad pathway acts at later timepoints and presumably it is responsible for the retention of transcriptional activation of the RhoB gene and by this way retention of the phenotype of actin cytoskeleton reorganization caused by TGFβ c) TGFβ treatment for 24hours results in obvious reorganization of actin cytoskeleton
(stress fibers) in HaCaT cells. The use of MEK inhibitor U0126 induced this reorganization presumably due to the small transcriptional upregulation of the RhoA gene caused by the MEK inhibitor. d) Overexpression of a constitutively active TGFβ receptor type I by the use of recombinant adenoviruses resulted in the upregulation of RhoB at the transcriptional and protein level in HepG2 cells e) A functional characterizaton of RhoB promoter showed that the region between nucleotides -78 and +19 is necessary and sufficient for transactivation of RhoB promoter by the Smad proteins. f) Overexpression of a constitutively active oncoprotein Ras (Ras ca) transactivates the RhoB promoter (-726 +86). This specific activation is reduced in mutants -227/+86 and -78/+86 of RhoB promoter. g) We didn’t observe synergistic interactions between the pathways of MAP kinases and Smad proteins in the regulation of the RhoB promoter.
In the second part of our study, we investigated the role of Sp1 in the transcriptional activation of RhoB gene. Our experimental data showed that Mithramycin, a drug that inhibit the binding of Sp1 to GC-rich motives in the promoters of genes, has a negative effect on the basal levels of transcription of RhoB gene. Overexression of Sp1 induces the activity of RhoB promoter. The RhoB promoter consists of three Sp1 binding sites (-660, -123 and -104).
In the third part of this study, we investigated the importance of p53 family in the transactivation of the RhoB promoter. We showed that among the members of this family, only p73 regulates the transcription of RhoB gene by activating its promoter. This observation supports previous observations showing that p53 family members have overlapping but distinct roles on target genes. The minimum region that is responsible for transactivation by p73 is -78 /+86. Finally, we observed synergistic interactions between p73 and Sp1 in transactivation of RhoB promoter.
The study of RhoB regulation at the transcriptional and protein level will provide important information regarding its function and may lead to a deeper understanding of its special functional characteristics. Studying the role of RhoB in actin cytoskeleton reorganization caused by TGFβ in addition to its known anticancer action, may lead to the discovery of novel tools for the treatment of diseases such as cancer.
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Μηχανισμοί μεταγραφικής ενεργοποίησης της μικρής GTPάσης RhoB
