Abstract
The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-β type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-β1 stimulation. RAC1B KO also increased TGF-β1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivation of the ALK5 kinase activity by dominant-negative interference or ATP-competitive inhibition rescued the cells from the RAC1B KD/KO-mediated increase in TGF-β1-induced cell migration, whereas the ectopic expression of kinase-active ALK5 mimicked this RAC1B KD/KO effect. We conclude that RAC1B downregulates the abundance of ALK5 and SMAD3 signaling, thereby attenuating TGF-β/SMAD3-driven cellular responses, such as growth inhibition and cell motility.
Highlights
related C3 botulinum toxin substrate 1B (RAC1B), and its more prominent isoform, Ras-related C3 botulinum toxin substrate 1 (RAC1), are encoded by the human RAC1 gene
Our published data suggest that RAC1B suppression of cell migration may involve downregulation of transforming growth factor (TGF)-β1-induced phosphorylation of SMAD3C [2], p38 MAPK, and extracellular signal-regulated kinase (ERK)1/2 MAPK [3], which are critical for TGF-β1-induced migration
Previous data obtained with Panc1 cells have shown that KD of RAC1B via a siRNA targeting exon 3b of RAC1 resulted in elevated levels of activin receptor-like kinase 5 (ALK5) mRNA [3]
Summary
RAC1B, and its more prominent isoform, Ras-related C3 botulinum toxin substrate 1 (RAC1), are encoded by the human RAC1 gene. RAC1B differs from RAC1 by in-frame insertion of exon 3b, encoding for 19 amino acids, resulting in a small GTPase with impaired enzymatic activity but an accelerated ability to exchange GDP to GTP [1]. Our RNAi-triggered knockdown (KD) analyses suggest that endogenous RAC1B and RAC1 suppress and promote, respectively, TGF-β1-dependent migration (chemokinesis) of “normal” and malignant pancreatic epithelial cells [2,3], as well as carcinoma-derived cell lines of the breast [4,5] and prostate (H.U., unpublished data). Our published data suggest that RAC1B suppression of cell migration may involve downregulation of TGF-β1-induced phosphorylation of SMAD3C [2], p38 MAPK (microtubule-associated protein kinase), and extracellular signal-regulated kinase (ERK)1/2 MAPK [3], which are critical for TGF-β1-induced migration. The mechanism(s) whereby RAC1B interferes with SMAD and MAPK activation are not known yet
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