TGF-ß/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells.

The TGF-ß and Hippo pathways are critical for liver size control, regeneration, and cancer progression. The transcriptional cofactor TAZ, also named WWTR1, is a downstream effector of Hippo pathway and plays a key role in the maintenance of liver physiological functions. However, the up-regulation of TAZ expression has been associated with liver cancer progression. Recent evidence shows crosstalk of TGF-ß and Hippo pathways, since TGF-ß modulates TAZ expression through different mechanisms in a cellular context-dependent manner but supposedly independent of SMADs. Here, we evaluate the molecular interplay between TGF-ß pathway and TAZ expression and observe that TGF-ß induces TAZ expression through SMAD canonical pathway in liver cancer HepG2 cells. Therefore, TAZ cofactor is a primary target of TGF-ß/SMAD-signaling, one of the pathways altered in liver cancer.

Hepatic C9 cells switch their behaviour in short or long exposure to soft substrates.

BACKGROUND INFORMATION: There have been several studies to understand the influence of stiffness of the culture substrates for different types of adherent cells. It is generally accepted that cell proliferation, spreading and focal adhesions increase with higher matrix stiffness. However, what remains unclear is whether this kind of cell behaviour may be reverted by culturing on soft substrates those cell lines that were originally selected or primed on stiff surfaces. RESULTS: Here, we studied the influence of substrate softness on proliferation, adhesion and morphology of the highly proliferative hepatic C9 cell line. We cultured C9 cells on soft and stiff polydimethylsiloxane (PDMS) substrates prepared with two different elastic moduli in the range of 10 and 200 kPa, respectively. Lower cell proliferation was observed on substrates with lower stiffness without affecting cell viability. The proliferation rate of C9 cell line along with extracellular growth-regulated kinase (ERK) phosphorylation was decreased on soft PDMS. Despite this cell line has been described as a hepatic epithelial cell, our characterisation demonstrated that the origin of C9 cells is uncertain, although clearly epithelial, with the expression of markers of several hepatic cells. Surprisingly, consecutive passages of C9 cells on soft PDMS did not alter this mesenchymal phenotype, vimentin expression did not decrease when culturing cells in soft substrates, even though the ERK phosphorylation levels eventually were increased after several passages on soft PDMS, triggering again an increase of cell proliferation. CONCLUSIONS AND SIGNIFICANCE: This study shows that the exposure of C9 cells to soft substrates promoted a decrease of cell proliferation rate, as reported for other types of cells on PDMS, whereas a much longer term exposure caused cells to adapt to softness after trained for several passages, reactivating proliferation. During this phenomenon, the morphology and phenotype of trained cells was modified accompanying the increase of cell proliferation rate contrary to the effect observed in short periods of cell culture. In contrast to previous reports, cell death was not observed during these experiments, discarding a cell selection mechanism and suggesting soft cell adaptation may be limited in time in C9 cells.

Fabrication of low-cost micropatterned polydimethyl-siloxane scaffolds to organise cells in a variety of two-dimensioanl biomimetic arrangements for lab-on-chip culture platforms.

We present the rapid-prototyping of type I collagen micropatterns on poly-dimethylsiloxane substrates for the biomimetic confinement of cells using the combination of a surface oxidation treatment and 3-aminopropyl triethoxysilane silanisation followed by glutaraldehyde crosslinking. The aim of surface treatment is to stabilise microcontact printing transfer of this natural extracellular matrix protein that usually wears out easily from poly-dimethylsiloxane, which is not suitable for biomimetic cell culture platforms and lab-on-chip applications. A low-cost CD-DVD laser was used to etch biomimetic micropatterns into acrylic sheets that were in turn replicated to poly-dimethylsiloxane slabs with the desired features. These stamps were finally inked with type I collagen for microcontact printing transfer on the culture substrates in a simple manner. Human hepatoma cells (HepG2) and rat primary hepatocytes, which do not adhere to bare poly-dimethylsiloxane, were successfully seeded and showed optimal adhesion and survival on simple protein micropatterns with a hepatic cord geometry in order to validate our technique. HepG2 cells also proliferated on the stamps. Soft and stiff poly-dimethylsiloxane layers were also tested to demonstrate that our cost-effective process is compatible with biomimetic organ-on-chip technology integrating tunable stiffness with a potential application to drug testing probes development where such cells are commonly used.