No abstract

Transforming growth factor (TGF)-β1 promotes progression of pancreatic ductal adenocarcinoma (PDAC) by enhancing epithelial-mesenchymal transition, cell migration/invasion, and metastasis, in part by cooperating with the small GTPase Rac1. Prompted by the observation of higher expression of Rac1b, an alternatively spliced Rac1 isoform, in pancreatic ductal epithelial cells and in patients with chronic pancreatitis vs. PDAC, as well as in long-time vs. short-time survivors among PDAC patients, we asked whether Rac1b might negatively affect TGF-β1 prometastatic function. Interestingly, the non-malignant pancreatic ductal epithelial cell line H6c7 exhibited a higher ratio of active Rac1b to total Rac1b than the TGF-β1-responsive PDAC cell lines Panc-1 and Colo357. Notably, siRNA-mediated silencing of Rac1b increased TGF-β1/Smad-dependent migratory activities in H6c7, Colo357, and Panc-1 cells, while ectopic overexpression of Rac1b in Panc-1 cells attenuated TGF-β1-induced cell motility. Depletion of Rac1b in Panc-1 cells enhanced TGF-β1/Smad-dependent expression of promoter-reporter genes and of the endogenous Slug gene. Moreover, Rac1b depletion resulted in a higher and more sustained C-terminal phosphorylation of Smad3 and Smad2, suggesting that Rac1b is involved in Smad2/3 dephosphorylation/inactivation. Since pharmacologic or siRNA-mediated inhibition of Smad3 but not Smad2 was able to alleviate the Rac1b siRNA effect on TGF-β1-induced cell migration, our results suggests that Rac1b inhibits TGF-β1-induced cell motility in pancreatic ductal epithelial cells by blocking the function of Smad3. Moreover, Rac1b may act as an endogenous inhibitor of Rac1 in TGF-β1-mediated migration and possibly metastasis. Hence, it could be exploited for diagnostic/prognostic purposes or even therapeutically in late-stage PDAC as an antimetastatic agent.

We evaluated regional electrical impedance (Z degree) at 2.5 and 100 kHz to separate intra- and extracellular fluid changes and correlated Z degree over the thorax (TI) to relative changes in the central blood volume (CBV) induced by head-up tilt. In nine experiments head-up tilt resulted in normotensive central hypovolaemia associated with a 3.7 +/- 0.4 Ohm (mean +/- SE) increase in TI100 kHz after 60 min. In 24 experiments pre-syncopal symptoms were induced after 43 +/- 2 min, when TI100 kHz had increased 4.2 +/- 0.2 Ohm. Head-up tilt instantly decreased the activity of technetium labelled erythrocytes (99Tcm) over the thorax by 24 +/- 2%, and increased 99Tcm over the thigh by 68 +/- 10% (P less than 0.01, n = 8) with no further changes during the sustained tilt. Haematocrite increased during head-up tilt from 43.1 +/- 0.3 to 47.9 +/- 0.6% (P less than 0.01, n = 8). Accordingly, the increase in TI (6.3 +/- 0.6 vs. 4.5 +/- 0.4 Ohm, n = 6) and the decrease in Z degree through one leg (7.2 +/- 1.2 vs. 2.8 +/- 0.5 Ohm, n = 6) at 2.5 kHz was more pronounced than at 100 kHz. Also the changes in TI were correlated to CBV as calculated from 99Tcm and haematocrite (r = 0.90, P less than 0.01). The results suggest that: (1) Hypovolaemic shock is associated with a faster increase of TI than normotensive head-up tilt. (2) Head-up tilt is characterized by an initial decrease in CBV followed by a further decrease in plasma volume, which eventually leads to hypovolaemic shock. (3) Blood volume changes during head-up tilt are reflected in regional Z degree.