title: Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.
abstract: Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.
authors.affiliation: Department of Dermatology, University Medical Center St. Radboud, Nijmegen, The Netherlands. a.pol@derma.azn.nl
authors.forename: Arno
authors.initials: A
authors.lastname: Pol
authors.forename: Fred
authors.initials: F
authors.lastname: van Ruissen
authors.forename: Joost
authors.initials: J
authors.lastname: Schalkwijk
mesh_terms: D001681:Biological Assay; D015152:Blotting, Northern; D018929:Cell Culture Techniques; D002460:Cell Line; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000302:isolation & purification; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics*; D011565:Psoriasis / Q000188:drug therapy* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012867:Skin / Q000254:growth & development; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011480:Protease Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins