title: Hiding inside? Intracellular expression of non-glycosylated c-kit protein in cardiac progenitor cells.
abstract: Cardiac progenitor cells including c-kit(+) cells and cardiosphere-derived cells (CDCs) play important roles in cardiac repair and regeneration. CDCs were reported to contain only small subpopulations of c-kit(+) cells and recent publications suggested that depletion of the c-kit(+) subpopulation of cells has no effect on regenerative properties of CDCs. However, our current study showed that the vast majority of CDCs from murine heart actually express c-kit, albeit, in an intracellular and non-glycosylated form. Immunostaining and flow cytometry showed that the fluorescent signal indicative of c-kit immunostaining significantly increased when cell membranes were permeabilized. Western blots further demonstrated that glycosylation of c-kit was increased during endothelial differentiation in a time dependent manner. Glycosylation inhibition by 1-deoxymannojirimycin hydrochloride (1-DMM) blocked c-kit glycosylation and reduced expression of endothelial cell markers such as Flk-1 and CD31 during differentiation. Pretreatment of these cells with a c-kit kinase inhibitor (imatinib mesylate) also attenuated Flk-1 and CD31 expression. These results suggest that c-kit glycosylation and its kinase activity are likely needed for these cells to differentiate into an endothelial lineage. In vivo, we found that intracellular c-kit expressing cells are located in the wall of cardiac blood vessels in mice subjected to myocardial infarction. In summary, our work demonstrated for the first time that c-kit is not only expressed in CDCs but may also directly participate in CDC differentiation into an endothelial lineage.
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA.
authors.forename: Huilin
authors.initials: H
authors.lastname: Shi
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Drummond
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Fan
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Haller
authors.affiliation: Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Jiang
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA.
authors.forename: Deepak
authors.initials: D
authors.lastname: Malhotra
authors.affiliation: Department of Medicine, Division of Cardiovascular Medicine, College of Medicine and Life Sciences, University of Toledo, Toledo, OH 43614, USA. Electronic address: Jiang.Tian@utoledo.edu.
authors.forename: Jiang
authors.initials: J
authors.lastname: Tian
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D002478:Cells, Cultured; D006031:Glycosylation; D000068877:Imatinib Mesylate / Q000494:pharmacology; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology; D009206:Myocardium / Q000166:cytology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D019408:Platelet Endothelial Cell Adhesion Molecule-1; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: C-kit; Cardiac progenitor cell; Endothelial differentiation; Glycosylation