ABSTRACT
Although the effects of low-intensity pulsed ultrasound (LIPUS) on diverse cell types have been fully studied, the functional role of LIPUS in keratinocytes remains poorly understood. This study aimed to investigate the effects of LIPUS on proliferation and migration of HaCaT cells as well as the regulatory mechanisms associated with signaling pathways. Human HaCaT cells were exposed or not to LIPUS, and cell proliferation and migration were measured by BrdU incorporation assay and Transwell assay, respectively. Expression of proteins associated with proliferation and migration was evaluated by western blot analysis. Expression of key kinases in the PI3K/AKT and JNK pathways was also evaluated by western blot analysis. Effects of LIPUS on the PI3K/AKT and JNK pathways, and whether LIPUS affected HaCaT cells via these two pathways were finally explored. When the parameter of LIPUS (number of cycles) was set at 300, cell viability was the highest after LIPUS stimulation. We then found that the percentage of BrdU positive cells was enhanced by LIPUS, along with up-regulation of cyclinD1, CDK6, CDK4, and VEGF. LIPUS promoted migration, as well as up-regulation of MMP-2 and MMP-9. Phosphorylation levels of key kinases in the PI3K/AKT and JNK pathways were increased by LIPUS. Inhibition of either PI3K/AKT pathway or JNK pathway attenuated effects of LIPUS on HaCaT cells, and co-inhibition of these two pathways showed augmented effects. LIPUS promoted proliferation and migration of HaCaT cells through activating the PI3K/AKT and JNK pathways.
Subject(s)
Keratinocytes/radiation effects , Cell Movement/radiation effects , Phosphatidylinositol 3-Kinases/radiation effects , MAP Kinase Signaling System/radiation effects , Cell Proliferation/radiation effects , Ultrasonic Waves , Bromodeoxyuridine , Cell Line, Transformed , Signal Transduction/radiation effects , Keratinocytes/metabolism , Up-Regulation , Cell Survival/radiation effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , Phosphatidylinositol 3-Kinases/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolismABSTRACT
Background & objectives: Ex vivo expansion of the limbal epithelial cells activates the nerve growth factor (NGF) mediated downstream signal transduction pathway. It is not clear as to which factors control the stemness of the corneal limbal stem cells, i.e., the maintenance of stem cell properties. It is likely that various signaling pathways are involved, including Notch, Wnt and NGF signaling, etc. In the present study, we investigated the activation of phosphoinositide-3-kinase (PI3K) pathway on cells cultured over the chitosan matrix, chitosan silver matrix, chitosan gold matrix, intact and denuded human amniotic membrane (HAM). Methods: Human limbal biopsies obtained from the cadaveric donor eyes were used in this study. The cells cultured over different substrate and observed for the activation of the downstream signaling molecules of PI3K/Akt/FKHRL1 pathway. Western blotting was done to prove the results. Results: The cells cultured over the intact HAM showed the activation of the downstream signaling molecules of PI3K/Akt/JNK pathway compared to the cells grown over other substrates. On inhibition of the PI3K activity there was absence of phosphorylation of downstream effectors in the limbal epithelial cells from the explant culture over the intact HAM. Interpretation & conclusions: The ex vivo expansion of human limbal epithelial progenitor cells on intact HAM was mediated by PI3K/Akt/FKHRL1 pathway, which is known to govern cell survival, and the mitogen-activated protein kinase (MAPK) pathway, known to control the cell mitosis.