title: Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.
abstract: Cholesterol is organized in distinctive liquid-ordered micro-domains within biological membranes called lipid rafts. These micro-domains direct multiple physiological functions in mammalian cells by modulating signaling processes. Recent findings suggest a role for lipid rafts in cellular processes in human keratinocytes such as early differentiation and apoptosis. However, research of lipid rafts is hindered by technological limitations in visualizing dynamic cholesterol organization in plasma membranes. This study addresses a real-time, non-invasive method for the long-term observation of cholesterol reorganization in plasma membranes. In addition, this study also addresses the dynamic process of cholesterol depletion and repletion in primary human keratinocytes. Cholesterol reorganization was measured by observed changes in cellular impedance. Disruption of lipid rafts with low concentrations of methyl-beta-cyclodextrin (MbetaCD) resulted in an increase in the proliferative capacity of keratinocytes, which was assessed using real-time proliferation curves and adenosine triphosphate (ATP)-based proliferation assays. Quantitative PCR showed a concomitant decrease in messenger RNA (mRNA) expression of the early differentiation markers keratins 1 and 10. Conversely, specific cholesterol reintegration led to a 4.5-fold increase in keratin 2 mRNA expression, a marker for late keratinocyte differentiation, whereas depletion resulted in a significant downregulation. These findings imply a strictly controlled mechanism for the regulation of membrane cholesterol composition in both early and terminal keratinocyte differentiation. The impedance-based method that this study addresses further enhances our understanding of how physiological processes in keratinocytes are controlled by membrane cholesterol.
authors.affiliation: Research and Development, Beiersdorf AG, Hamburg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Spörl
authors.forename: Minetta
authors.initials: M
authors.lastname: Wunderskirchner
authors.forename: Oliver
authors.initials: O
authors.lastname: Ullrich
authors.forename: Gerrit
authors.initials: G
authors.lastname: Bömke
authors.forename: Ute
authors.initials: U
authors.lastname: Breitenbach
authors.forename: Thomas
authors.initials: T
authors.lastname: Blatt
authors.forename: Horst
authors.initials: H
authors.lastname: Wenck
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Wittern
authors.forename: Annika
authors.initials: A
authors.lastname: Schrader
mesh_terms: D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D017097:Electric Impedance; D000078404:Epidermal Cells; D005372:Filipin / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D053556:Keratin-1 / Q000235:genetics; D053550:Keratin-10 / Q000235:genetics; D053557:Keratin-2 / Q000235:genetics; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C491616:KRT1 protein, human; C507493:KRT10 protein, human; D053556:Keratin-1; D053557:Keratin-2; D012333:RNA, Messenger; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D053550:Keratin-10; D005372:Filipin; D002784:Cholesterol; D002118:Calcium