Three-Dimensional Invasion of Epithelial–Mesenchymal Transition–Positive Human Peritoneal Mesothelial Cells into Collagen Gel is Promoted by the Concentration Gradient of Fibronectin

BackgroundIn long-term peritoneal dialysis, myofibroblast-like cells found in the interstitium of the peritoneum are assumed to be a transformed type of mesothelial cell—epithelial-mesenchymal transition-positive [EMT(+)] human peritoneal mesothelial cells (HPMCs)—because they express a mesothelial marker, cytokeratin. However, no direct evidence about how these cells are able to invade from the mesothelium has yet been obtained.AimIn this study, we aimed to verify whether EMT(+) HPMCs would, in vitro, invade three-dimensionally along certain chemotactic factors.MethodsWe used reverse-transcriptase polymerase chain reaction to measure expression of Snail, E-cadherin, α5-integrin, and matrix metalloproteinase 2 (MMP2) messenger RNA (mRNA) in HPMCs exposed to 10 ng/mL transforming growth factor β1 (TGFβ1) and how that expression corresponds to cell motility, as represented by a video movie. We used the Transwell (12 μm pore diameter: Sigma-Aldrich, Tokyo, Japan) to construct a three-dimensional (3D) cell migration chamber. In the lower chamber, a concentration gradient of fibronectin (FN) or albumin(Alb) was formed in 0.1% type I collagen by diffusion ( C0= 22 nmol/L; concentration gradient: C / C0= 0.7). All cells beneath the membrane were counted 72 hours after 5x104EMT(+) HPMCs (HPMCs after a 48-hour exposure to 10 ng/mL TGFβ1) had been spread in the upper chamber.ResultsAfter 72 hours, the increased motility of HPMCs resulting from their exposure to 10 ng/mL TGFβ1 had returned to baseline, but they retained an elongated morphology. Expression of Snail and MMP2 mRNA reached maximum at 24 hours. Expression of E-cadherin declined, and expression of α5-integrin increased continuously. In the 3D invasion study, significantly enhanced invasion by EMT(+) but not EMT(-) HPMCs was clearly seen in the presence of a FN concentration gradient ( p < 0.01), although invasion by EMT(+) and EMT(-) HPMCs in the absence of a FN concentration gradient was not statistically significantly different. Compared with the EMT(+) control (no concentration gradient), invasion by EMT(+) HPMCs was 2.1 ± 0.5 times (p < 0.05) and 1.4 ± 0.4 times (p = nonsignificant) higher along the FN and Alb concentration gradients respectively. Increased invasion along the FN concentration gradient was significantly inhibited (p < 0.05) when the HPMCs were pre-incubated with 5 μg/mL RGDS (a blocker for α5-integrin to FN).ConclusionsWe conclude that EMT(+) HPMCs invade collagen gel along the FN concentration gradient because of specific binding to RGDS receptors, which bind integrins such as α5-integrin, upregulating invasion-related gene expression associated with synthesis of the cytoskeleton protein α smooth muscle actin.