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American Journal of Pathology, Vol 145, 561-573, Copyright © 1994 by American Society for Investigative Pathology

REGULAR ARTICLES

Expression of serine proteinases and metalloproteinases in organ- cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion

J Varani, B Burmeister, RG Sitrin, SB Shollenberger, DR Inman, SE Fligiel, DF Gibbs and K Johnson
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.

Neonatal human foreskin obtained at circumcision was cut into 2 x 2-mm pieces and placed in organ culture. Culture medium consisted of a serum- free, growth factor-free basal medium containing either 0.15 mmol/L Ca2+ or 1.4 mmol/L Ca2+. Some cultures were left as control, whereas others were treated with 3 mumol/L all-trans retinoic acid (RA). In the presence of RA, epidermal cohesion was disrupted and the upper layers separated from the viable epidermis beneath. This effect was observed under both low Ca2+ and high Ca2+ conditions. At 2-day intervals, culture fluids were collected and analyzed for serine and metalloproteinase activities. Serine proteinase activity was detected in the culture fluids and virtually all of the detected activity was dependent on the presence of plasminogen. Activity was elevated in the RA-treated tissues and this was due to increased amounts of both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Elastase and cathepsin G were not detected in either control or RA-treated cultures. Increased plasminogen activator levels were also detected in RA-treated keratinocytes and fibroblasts in monolayer culture. Significant amounts of t-PA (though not u-PA) were found in fibroblast culture fluids, whereas both t-PA and u-PA were detected in culture fluids from keratinocytes. Metalloproteinase activity was also detected in the culture fluids of control and RA- treated tissues but in contrast to plasminogen activator, metalloproteinase activity decreased in the presence of RA. Casein and gelatin zymographic studies indicated the presence of both 92- and 72- kd gelatinases and stromelysin-1 and suggested that the decreased activity was primarily due to reduction in the 92- and 72-kd gelatinases. When serine proteinase inhibitors (aprotinin and soybean trypsin inhibitor) were included in the culture medium throughout the incubation period, epidermal discohesion was reduced. A metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-2, did not have this effect. Taken together, these data show that a number of proteolytic enzymes are produced during organ culture of human skin. They suggest that these proteases may influence the structural integrity of the tissue.

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