Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhib itor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain pr imarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an un known cytoplasmic proteinase. During interaction with the target proteinase , cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibod y specific for PAI-2r, we demonstrate here that PAI-2r is present in kerati nocytes of the granular and basal layers of normal human epidermis. In addi tion, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiatin g cells. These data provide evidence for the presence of an endogenous, ker atinocyte-derived proteinase that constitutively cleaves intracellular PAI- 2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this protei nase may reflect specific intracellular action of PAI-2 in normal cells. Fi nally, we demonstrate that a commercially available anti-PAI-2 monoclonal a ntibody (#3750, American Diagnostica, Greenwich, CT), under native experime ntal conditions, preferentially recognizes the uncleaved, active form of PA I-2 and does not efficiently detect PAI-2r. (C) 2000 Wiley-Liss, Inc.