A Partial Catalog of Proteins Secreted by Epidermal Keratinocytes in Culture

Proteins secreted by epidermal keratinocytes are known to engage in functions other than those directly associated with barrier formation. We have used a previously published culture model to collect proteins secreted by adult human epidermal keratinocytes. Electrophoresis and microsequencing allowed us to identify 20 proteins. The list of proteins includes those known to be produced by keratinocytes (β-2 microglobulin, βIG-H3, calgranulin A, cathepsin B and D, E-cadherin, gelatinase B, gelsolin, interstitial collagenase, laminin B2t, plasminogen activator inhibitor-1, protein 14–3-3ε, SCC antigen, stratifin, and translationally controlled tumor protein) as well as those not previously known to be secreted by keratinocytes (epididymis secretory protein, maspin, and anti-neoplastic urinary protein). In addition, two proteins were identified that are not known to be secreted (glutathione-S-transferase and heat shock protein 27/28 kDa). The varied nature of the proteins identified suggests that epidermal keratinocytes have physiologic functions that have yet to be identified

Epithelium-specific Response of Cultured Keratinocytes to Infection with Adenovirus Type 2

Adenoviruses are pathogenic for certain stratified squamous epithelia. The sites most frequently involved are the upper respiratory tract and oropharynx. Adenovirus infections of the epidermis are quite rare. We examined the virus-cell interactions of adenovirus type 2 (Ad2) and cultured human keratinocytes grown from a variety of body sites. Our intent was to explore the nature of the apparent epithelium-specific susceptibility to Ad2. In brief, we found that in vitro viral susceptibility of the keratinocytes could be reliably predicted based on whether the cells originated from an epidermal or oropharyngeal surface. Ad2 proceeded through a complete vegetative cycle when used to infect cultured keratinocytes from oropharyngeal sites (e.g., gingiva and soft palate). In contrast, Ad2 infection was severely restricted in keratinocytes from epidermal sites (e.g., foreskin, abdomen, and buttock). These results demonstrate that the in vitro response to infection with Ad2 reflects in vivo tissue-specific susceptibility. In vivo, cervical epithelium is rarely infected with Ad2 and yet in culture, cervical keratinocytes were fully permissive for Ad2 replication. We propose that the permissive or nonpermissive response to Ad2 may be regulated by a particular aspect of cell phenotype. Because the permissive responses seen in this study were all generated in keratinocytes from mucosal sites, it is possible the in vitro response to Ad2 reflects inherent differences between mucosal and epidermal keratinocytes

Retrovirus-Mediated Transduction of Cultured Epidermal Keratinocytes

Retrovirus-mediated gene transfer is an efficient means of introducing and expressing exogenous gene(s) in many cell types including keratinocytes. However, parameters of transduction and gene expression have not been systematically analyzed for keratinocytes. To carry out such a study we have transduced cultures of newborn foreskin cells with retroviral vectors that encode the genes for neomycin resistance (neor) and for beta-galactosidase (B-gal). The neor gene is a dominant selectable marker and the B-gal gene encodes a histochemically detectable product. Our key findings are the following: 1) all keratinocytes that form colonies can be successfully transduced at a viral titer greater than 5 × 106 colony-forming units/ml; 2) transduction is effected by integration of a single copy of retroviral DNA; 3) transduced cells are not at a growth disadvantage and, in fact, single clones of transduced keratinocytes can be expanded to yield over 109 cells, suggesting that stem cells are transduced; 4) whereas most transduced colonies exhibit B-gal staining in a high percentage of constituent cells, some colonies had a mosaic or sectored staining pattern; 5) expression of the non-selectable B-gal gene was somewhat greater in differentiated cells of the culture as compared to nondifferentiated precursors. The ability to transduce stem cells at a high efficiency and to follow expression of transduced genes in clonal progeny will allow lineage mapping in stratified epithelial tissues

Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin

Continuous renewal of the epidermis and its appendages throughout life depends on the proliferation of a distinct population of cells called stem cells. We have used in situ retrovirus-mediated gene transfer to genetically mark cutaneous epithelial stem cells of adolescent mice, and have followed the fate of the marked progeny after at least 37 epidermal turnovers and five cycles of depilation-induced hair growth. Histological examination of serial sections of labeled pilosebaceous units demonstrated a complex cell lineage. In most instances, labeled cells were confined to one or more follicular compartments or solely to sebaceous glands. Labeled keratinocytes in interfollicular epidermis were confined to distinct columnar units representing epidermal proliferative units. The contribution of hair follicles to the epidermis was limited to a small rim of epidermis at the margin of the follicle, indicating that long term maintenance of interfollicular epidermis was independent of follicle-derived cells. Our results indicate the presence of multiple stem cells in cutaneous epithelium, some with restricted lineages in the absence of major injury