|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Thiols stabilize cobblestone morphology of cultured mesothelial cells
Stephen D Bird*1, Michael Legge† and Robert J. Walker*
*Department of Medical and Surgical Sciences, University of Otago, Dunedin School of Medicine, Dunedin, New Zealand, and †Department of Biochemistry, University of Otago, Dunedin School of Medicine, Dunedin, New Zealand
Cellular thiols including GSH (glutathione) and l-Cys (l-cysteine) are essential for cell signalling, growth and differentiation. l-Cys is derived from the extracellular thiol pool and is the rate-limiting compound for intracellular GSH biosynthesis. The present study investigated the effect of thiol-supplemented medium on cell growth, phenotype and total GSH of cultured hPMCs (human peritoneal mesothelial cells). Cells were cultured in medium M199 supplemented with 2% serum, with ‘plus’ or without ‘minus’ l-Cys and compared with medium supplemented with either β-ME (β-mercaptoethanol) (0.25 mmol/l) or the receptor tyrosine kinase ligand EGF (epidermal growth factor, 100 ng/ml). β-ME produced a disproportionate increase in total GSH compared with l-Cys and other thiols tested [(procysteine (2-oxothiazolidine-4-carboxylic acid) or NAC (N-acetyl-l-cysteine)], while growth and morphology were identical. Cell behaviour of primary hPMCs is characterized by the transition of fibroblastoid to cobblestone morphology during early passage. l-Cys and β-ME promoted a rapid MET (mesenchymal-to-epithelial transition) within 3 days of culture, confirmed by the presence of cobblestone cells, intact organelles, abundant microvilli, primary cilia and cortical actin. In contrast, EGF produced a biphasic response consisting of delayed growth and retention of a fibroblastoid morphology. During a rapid log phase of growth, MET was accompanied by rapid catch-up growth. Thiols may stabilize the epithelial phenotype by engaging redox-sensitive receptors and transcription factors that modulate differentiation. These data may benefit researchers working on thiol-mediated cell differentiation and strategies to regenerate damage to serosal membranes.
Key words: β-mercaptoethanol, actin, biocompatibility, EGF, extracellular thiol, glutathione, mesenchymal-to-epithelial transition, oxidative stress, primary cilium, serum depletion, thiol redox
Abbreviations: β-ME, β-mercaptoethanol, Ca2+i, intracellular calcium, EGF, epidermal growth factor, FBS, fetal bovine serum, GSH, glutathione, GSSG, glutathione disulfide, hPMC, human peritoneal mesothelial cell, KRS, Krebs–Ringer solution, l-Cys, l-cysteine, l-CySS, l-cystine, MET, mesenchymal-to-epithelial transition, MVB, multivesicular bodies, NAC, N-acetyl-l-cysteine, PD, peritoneal dialysis, procysteine, 2-oxothiazolidine-4-carboxylic acid, ROS, reactive oxygen species
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Received 18 August 2010/24 February 2011; accepted 10 March 2011
Published as Cell Biology International Immediate Publication 10 March 2011, doi:10.1042/CBI20100593
© The Author(s) Journal compilation © 2011 Portland Press Limited