| Abstract |
Rho proteins are characterized as molecular switches that control many
cellular processes including actin and microtubule cytoskeleton organization, cell
division, motility, cell adhesion, vesicular trafficking, phagocytosis and transcriptional
regulation. 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.
The purpose of the present study was to investigate the mechanism of
transcriptional induction of the small GTPase RhoB gene by the Transforming
Growth Factor β (TGFβ-1) signaling pathway and the role of this regulation in TGFβ-
1-induced cell responses such as cell migration, epithelial to mesenchymal transition
(EMT) and cell growth.
We found that TGFβ-1 induces a rapid and sustained increase in the mRNA
and protein levels of the RhoB gene in various cell lines including human HaCaT
keratinocytes, human hepatoblastoma HepG2 cells, human prostate cancer DU145
and PC3 cells and in mouse Swiss3T3 fibroblasts. We showed that the RhoB gene is
a direct transcriptional target of TGFβ-1 in HaCaT cells. We found that this regulation
is specific for the RhoB gene and is facilitated by the simultaneous activation of
cytoplasmic Smad and MEK/ERK pathways.The early activation of the MEK/ERK
pathway is required for the recruitment of Smad3 to a novel, non-classical, Smad
binding element in the proximal RhoB promoter, in a p53-dependent manner. This
element is overlapping with a CCAAT box that constitutively binds Nuclear Factor Y.
Mutagenesis of this site abolished the Smad and NF-Y mediated transactivation of
the RhoB promoter.
Using siRNA-mediated silencing, inhibition of GTPase activity by dominant
negative forms and adenovirus-mediated gene transfer we established that RhoB
plays an important role in TGFβ-1-induced cell migration of HaCaT, DU145 and PC3
cells. Finally, we revealed a novel mechanism of cross-talk between the classical
TGFβ-1 ⁄Smad pathway and Rho GTPases in which RhoB participates in an
autoinhibitory loop of the TGFβ-1/Smad pathway. This mechanism explains the
negative role of RhoB in the TGFβ-1-induced epithelial to mesenchymal transition
and cell growth arrest. In conclusion, the specific induction of RhoB by TGF-β and its
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positive or negative role in TGF-β-induced cell responses indicate that RhoB could
be a target for therapeutic intervention in malignant invasive tumors.
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Μελέτη του μηχανισμού μεταγραφικής ρύθμισης του γονιδίου της μικρής GTPAΣΗΣ ΡΗΟΒ απο το μονοπάτι του μετασχηματίζοντα Αυξητικού παράγοντα β (TGFβ-1) και ο ρόλος της στις TGFβ-1- επαγώμενες αποκρίσεις του κυττάρου
