title: Platelet derived growth factor (PDGF) responsive epidermis formed from human keratinocytes transduced with the PDGF beta receptor gene.
abstract: Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes are not known to express any member of the platelet-derived growth factor receptor family. In order to study if epidermis may be genetically transformed to a platelet-derived growth factor sensitive compartment we aimed to introduce the gene encoding human platelet-derived growth factor receptor beta (PDGF beta R) into epidermal keratinocytes using a retrovirus-derived vector. Successful gene transfer to primary cells was confirmed by immunofluorescence staining, southern blotting, and ligand-induced receptor autophosphorylation. By culturing a mixture of PDGF beta R-transduced and unmodified keratinocytes at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue cultured for 6 days and examined by immunofluorescence microscopy was shown to contain PDGF beta R-expressing keratinocytes distributed in all layers of living epidermis. By continued tissue culture in serum-containing medium, the epidermis became increasingly cornified although receptor-positive cells were still observed within the viable basal compartment. Stimulation of PDGF beta R-transduced epidermis with recombinant platelet-derived growth factor BB had a mitogenic effect as reflected by an increased frequency of Ki-67 positive keratinocytes. The study demonstrates that transgene expression of human PDGF beta R can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation of such gene-modified skin equivalent enhances cell proliferation. In perspective, viral PDGF beta R gene transfer to keratinocytes may be a useful approach in studies of receptor tyrosine kinase mediated skin repair and epithelialization.
authors.affiliation: Department of Medical Sciences, Dermatology and Venereology, Uppsala University Hospital, Uppsala, Sweden. ola.rollman@medsci.uu.se
authors.forename: Ola
authors.initials: O
authors.lastname: Rollman
authors.forename: Uffe B
authors.initials: UB
authors.lastname: Jensen
authors.forename: Arne
authors.initials: A
authors.lastname: Ostman
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.forename: Sigrún M
authors.initials: SM
authors.lastname: Gústafsdóttir
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Jensen
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D015139:Blotting, Southern; D002455:Cell Division; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004817:Epidermis / Q000378:metabolism*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism*; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D049452:Green Fluorescent Proteins; D000077214:Becaplermin; D019000:Phosphotyrosine; D020797:Receptor, Platelet-Derived Growth Factor beta