Enhancement Of The Responsiveness Of TRPV Channels On HepG2 Cells With Micropillar Arrayed Topography

Polydimethylsiloxane(PDMS) micropillar arrayed topographic substrates were fabricated, with dimensions of 4 and 10μm in nominal pillar diameter, 4 and 7μm in spacing and 4μm in height, to investigate the expression and responsiveness of transient receptor potential(TRP) V1 and TRPV4 channels of Hep G2 cells cultured on the substrates.Our experiments showed that the morphological spreading and degree of polarization of Hep G2 cells changed significantly on the topographic substrates, as compared to cells on flat PDMS substrates. Quantitative real-time PCR(q RT-PCR) analysis revealed that the m RNA levels of TRPV1 and TRPV4 were significantly up-regulated in the cells grown on all four topographic substrates, when compared with the cells grown on the flat substrates. In the same dimensions of 4 or 10μm, the topographic substrates with smaller spacing(4μm) were related to higher spreading areas and lower m RNA levels of TRPV1 and TRPV4, when compared with the cells grown on the topographic substrates with larger spacing(7μm). In the same spacing of 4 or 7μm, the topographic substrates with larger dimensions(10μm) were related to higher spreading areas and higher m RNA levels of TRPV1 and TRPV4, when compared with the cells grown on the topographic substrates with smaller dimensions(4μm). The presence of TRPV1 and TRPV4 proteins in Hep G2 cells was confirmed with Western blotting and similar up-regulation of these two channel proteins by the substrate topography also revealed by stronger immunofluorescence staining. Subsequently, the channel responsiveness of TRPV1 and TRPV4 was quantified by using the calcium inflow-responding magnitudes and percentages of cells having a defined responding magnitude upon stimulation by the channel agonists capsaicin and 4-α-phorbol- 12, 13-didecanoate(4αPDD), respectively. Herein, Calcium Green-1 as a fluorescent indicator was employed in its dynamic assessment by confocal laser scanning microscopy. The results displayed that upon stimulation by the channel agonist, the calcium influx through TRPV1 exhibits a dynamic characteristic of rapid desensitization with a transient within 25 seconds, and either the relative fluorescence responding amplitudes or the percentages of responsive cells on the topographic substrates are greater than those observed from the cells on the flat substrates. By contrast, stimulation by the TRPV4 agonist caused a calcium response with much slower recovery within 5 minutes, and both the relative fluorescence responding amplitudes and the percentages of responsive cells on the topographic substrates are higher than those of the cells on the flat substrates.Collectively, these data indicate that the TRPV-mediated ion signaling elicits an important role in the cellular phenotypic and functional regulation by the substrate topography.