Suppression of SCARA5 by Snail1 is essential for EMT-associated cell migration of A549 cells.

Accumulating evidence indicates that epithelial-to-mesenchymal transition (EMT) might be a key event for cancer progression. The upregulation of Snail1, one of the most extensively studied EMT regulators, has been implicated in cancer metastasis, but the underlying mechanisms remain unclear. This study aims to identify that Snail1 targets regulating EMT-associated cancer cell migration. Human lung carcinoma A549 cells were treated with transforming growth factor beta 1 (TGF-ß1), and EMT-associated phenotypic and functional alterations were monitored. TGF-ß1 induced typical EMT-like morphological changes, 'cadherin switching' and cell migration in A549 cells. TGF-ß1 stimulation induced rapid and persistent upregulation of Snail1. Moreover, Snail1 upregulation was required for EMT-associated cell migration. Several metastasis suppressors with putative Snail1-binding sites in their promoters were dramatically repressed in A549 cells during TGF-ß1-induced EMT. Gain- and loss-of Snail1 function experiments demonstrated that scavenger receptor class A member 5 (SCARA5) was negatively regulated by Snail1. Importantly, SCARA5 downregulation was essential for EMT-induced migration in A549 cells. The chromatin immunoprecipitation assay revealed that Snail1 could bind to the E-box elements in SCARA5 promoter, implying that SCARA5 is a direct Snail1 target modulating cancer cell mobility during EMT. In addition, we showed that DNA methyltransferase 1 was physically associated with Snail1 to silence SCARA5 expression with an unidentified DNA methylation-independent mechanism, suggesting the complexity of Snail1-mediated epigenetic regulation. Collectively, our data demonstrated that EMT-regulator Snail1 suppresses the expression of SCARA5 to promote cancer progression, highlighting the possibility to target Snail1 and SCARA5 for cancer treatment.

Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.

BACKGROUND & AIMS: We report a novel approach to study biliary water, bile acid, and HCO(3)(-) transport: the microperfusion of intrahepatic bile duct units (IBDUs) isolated from normal rat liver. METHODS: To study water transport, IBDUs were perfused in vitro with a membrane-impermeant fluorescent volume marker, fluorescein sulfonate; net water movement (J(v)) and osmotic water permeability (P(f)) were then calculated. To study solute transport, IBDUs were perfused with taurocholic acid (TCA) and bile acid uptake was calculated from the concentrations of TCA in the perfused and collected solutions. To study ion transport, IBDUs were perfused with the cell-impermeant pH-sensitive dye BCECF dextran; luminal pH was determined from fluorescence excitation ratios. RESULTS: When inward (secretory) or outward (absorptive) osmotic gradients were established across IBDUs, water movement was observed from bath to lumen (i.e., secretion) and from lumen to bath (i.e., absorption). The perfused IBDUs absorbed TCA in a saturable, sodium-dependent manner; in addition, TCA absorption was blocked in a dose-dependent fashion by S0960, a specific inhibitor of the Na(+)/bile acid cotransporter. Addition of forskolin to HCO(3)(-)-containing (but not HCO(3)(-)-free) bath buffer resulted in lumen alkalinization reflecting HCO(3)(-) transport into the lumen of perfused IBDUs. CONCLUSIONS: The results provide direct functional evidence that intrahepatic bile ducts both secrete and absorb water in response to osmotic gradients, actively absorb bile acid, and transport HCO(3)(-).