Abstract
Prompted by earlier findings that the Rac1-related isoform Rac1b inhibits transforming growth factor (TGF)-β1-induced canonical Smad signalling, we studied here whether Rac1b also impacts TGF-β1-dependent non-Smad signalling such as the MKK6-p38 and MEK-ERK mitogen-activated protein kinase (MAPK) pathways and epithelial-mesenchymal transition (EMT). Transient depletion of Rac1b protein in pancreatic cancer cells by RNA interference increased the extent and duration of TGF-β1-induced phosphorylation of p38 MAPK in a Smad4-independent manner. Rac1b depletion also strongly increased basal ERK activation - independent of the kinase function of the TGF-β type I receptor ALK5 - and sensitised cells towards further upregulation of phospho-ERK levels by TGF-β1, while ectopic overexpression of Rac1b had the reverse effect. Rac1b depletion increased an EMT phenotype as evidenced by cell morphology, gene expression of EMT markers, cell migration and growth inhibition. Inhibition of MKK6-p38 or MEK-ERK signalling partially relieved the Rac1b depletion-dependent increase in TGF-β1-induced gene expression and cell migration. Rac1b depletion also enhanced TGF-β1 autoinduction of crucial TGF-β pathway components and decreased that of TGF-β pathway inhibitors. Our results show that Rac1b antagonises TGF-β1-dependent EMT by inhibiting MKK6-p38 and MEK-ERK signalling and by controlling gene expression in a way that favors attenuation of TGF-β signalling.
Highlights
Unequivocally to the absence of Rac[1]
As far as Rac[1] is concerned, we have shown earlier that Rac[1] promotes transforming growth factor (TGF)-β1 signalling in PDAC-derived cell lines towards a pro-metastatic outcome by enhancing TGF-β1-induced Smad[2] activation, epithelial-mesenchymal transition (EMT), and random cell migration and invasion[8]
We have previously shown that TGF-β1-induced activation of both Smad[2] and p38 mitogen-activated protein kinase (MAPK) is Rac1-dependent in PDAC-derived cells[8,29]
Summary
Unequivocally to the absence of Rac[1]. A solution to this dilemma would be a selective depletion of exclusively one of both isoforms, such data are not yet available. In light of the previously observed (negative) regulation of TGF-β1-induced Smad2/3 activation and random cell migration in PDAC-derived cells[8,9], it is conceivable that Rac1b affects activity of TGF-β1-dependent MKK6-p38 and/or MEK-ERK signalling as well as adoption of a mesenchymal phenotype. It should be mentioned, that due to the Smad- and ALK5 serine/threonine kinase-independent activation of p38 and ERK1/2, respectively, the effects of Rac1b on the Smad signalling pathway are not predictive of the effects of Rac1b on either the MKK6-p38 or the MEK-ERK signalling pathway. In some experiments we included non-tumourigenic cells of pancreatic and non-pancreatic origin to evaluate whether the observed effects were tumour and tissue-specific, respectively
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