Abstract
Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.
Highlights
Autoantibodies to collagen XVII perturb cell adhesion and lead to epidermal-dermal separation and skin blistering [3]
COS-7 cells were co-transfected with cDNA encoding collagen XVII and placental alkaline phosphatase (PLAP) or human transferrin receptor (HTrR), respectively, and incubated with primary and secondary antibodies for 60 min at 12 °C to minimize the metabolic activity of the cells
These findings indicate that a major portion of collagen XVII molecules are incorporated into lipid raft microdomains in the cell plasma membrane
Summary
Cell Cultures and Transient Transfections—HaCaT keratinocytes were cultured in serum-free keratinocyte growth medium supplemented with bovine pituitary extract and epidermal growth factor (Invitrogen) as described previously [3]. For determination of the inhibition profile of cholesterol-dependent sheddases, HaCaT keratinocytes were treated with 10 mM MCD in combination with the well known class-specific proteinase inhibitors E-64 (100 M), pepstatin A (100 M; Sigma), or 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF or Pefabloc) (1 mM; Merck) for 60 min. For analysis of ADAMs, HaCaT keratinocytes and COS-7 cells transfected with collagen XVII cDNA were treated with 10 mM MCD in combination with 35 M BB3241 (British Biotech Ltd.) or 35 M TNF-␣ protease inhibitor (Calbiochem) in serum-free Dulbecco’s modified Eagle’s medium for 60 min. As a negative control to demonstrate that the patching was antibody-induced, co-transfected COS-7 cells on coverslips were fixed with 2% paraformaldehyde and stained with the same set of primary antibodies in the respective dilutions In this case the proteins were randomly distributed over the plasma membrane
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