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 (beta-2 microglobulin, betaIG-H3, calgranulin A, cathepsin B and D, E-cadherin, gelatinase B, gelsolin, interstitial collagenase, laminin B2t, plasminogen activator inhibitor-1, protein 14-3-3epsilon, 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.

Epidermis as a secretory tissue: an in vitro tissue model to study keratinocyte secretion.

In addition to protective functions, keratinocytes secrete a variety of effector molecules that may have local or distant effects. To explore the secretory activity of keratinocytes we have developed a two-chamber culture model in which a fully differentiated stratified epithelium is present in the upper chamber and secreted protein is collected in a lower chamber. A collagen matrix is not used and during collection of secreted protein, 3T3 feeder cells are absent. Keratinocytes secrete protein at the rate of 0.67 microgram/h/10(6) cells, encompassing a molecular weight range from several kD to over 180 kD. Secretion is an unexplored area of keratinocyte biology and this model will allow investigation of this activity.

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 x 10(6) 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 10(9) 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.