Cell Confluence Regulates Hepatocyte Growth Factor-Stimulated Cell Morphogenesis in a β-Catenin-Dependent Manner

Abstract

Following organ injury, morphogenic epithelial responses can vary depending on local cell density. In the present study, the role of cell confluence in determining the responsiveness of renal epithelial cells to the dedifferentiating morphogenic signals of hepatocyte growth factor (HGF) was examined. Increasing confluence resulted in a greater tendency of cells to organize into epithelial tubes and a significant decrease in migratory responsiveness to HGF. Analysis of downstream signaling revealed that the HGF receptor c-Met was equally activated in confluent and nonconfluent cells following HGF stimulation but that phosphoinositide 3-kinasedependent activation of Akt and Rac were selectively diminished in confluent cells. In nonconfluent cells treated with HGF, the high level of Akt activation resulted in inhibitory phosphorylation of glycogen synthase kinase 3␤ (GSK-3␤) and increased ␤-catenin nuclear signaling. In contrast, confluent cells, in which HGFstimulated Akt activation was diminished, displayed less inhibitory phosphorylation of GSK-3␤ and less nuclear signaling by ␤-catenin. Overexpression of ␤-catenin (SA), which cannot be phosphorylated by GSK-3␤ and targeted for ubiquitination, significantly increased migration in fully confluent cells. Thus, cells maintained at high confluence selectively downregulate signaling events such as Rac activation and ␤-catenindependent transcription that would otherwise promote cell dedifferentiation and migration.

Hepatocyte Growth Factor (Hgf) Stimulates Low Density Lipoprotein Receptor-related Protein (Lrp) 5/6 Phosphorylation and Promotes Canonical Wnt Signaling

“…These findings, coupled with our previous demonstration that Hgf-mediated inhibition of cytosolic Gsk3 occurs only in subconfluent cells (10), suggest that Hgf/Met signaling specifically transactivates Wnt signaling in low confluency, de-differentiated epithelial cells, reminiscent of sub-lethally injured cells after AKI. This pathway is apparently suppressed as cells become more differentiated and confluent, resembling the healthy, uninjured epithelial cells of the renal tubule.…”

“…Hgf induces Akt-dependent phosphorylation of Gsk3␤ at Ser-9 and Gsk3␣ at Ser-21, resulting in a conformational change that promotes the N terminus to become a pseudo-substrate that blocks the phospho-binding site and inhibits Gsk3 kinase activity (10,40). In the current study, we show that stimulation of cells with Hgf results in the selective exclusion of this Ser-9 phosphorylated pool of Gsk3␤ from association with Lrp5/6, even though there is a marked increase in Ser-9-phosphorylated Gsk3␤ at the whole cell level.…”

“…Hgf-stimulated Phosphorylation of Lrp5/6 Is Dependent on Cell Confluency-We have previously shown that both Hgf signaling and expression of epithelial differentiation markers are regulated by cell confluency (10,26). To determine the impact of confluency on Hgf-stimulated phosphorylation of Lrp5/6, MPT cells were plated at low, medium, and high confluency (representative images in Fig.…”

“…We have previously demonstrated that Hgf stimulation of subconfluent renal TECs leads to Akt-mediated inhibition of Gsk3 (10). In the current study, we further investigated the cross-talk between these pathways and its significance.…”

Differential expression of Snail1 transcription factor and Snail1-related genes in alveolar and embryonal rhabdomyosarcoma subtypes.

“…The expression of Snail1 is higher in alveolar subtype in comparison to embryonal in the patients' samples and this finding was confirmed using established ARMS and ERMS cell lines both on RNA and protein level. [21,24] and, the same, inactivation of β-Catenin degradation complex (1, 2). β-Catenin, freed from destructive influence of its degradation complex (3), is transported to the nucleus (4), where, acting through TCF and LEF nuclear proteins it can work as a transcription factor.…”