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 inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily 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 unknown 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 antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.
Risse
B C
BC
Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104-6142, USA.
Chung
N M
NM
Baker
M S
MS
Jensen
P J
PJ
D000906:Antibodies / Q000276:immunology; D003902:Detergents / Q000494:pharmacology; D004351:Drug Resistance; D010450:Endopeptidases / Q000378:metabolism; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D008968:Molecular Conformation; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D012016:Reference Values
D000906:Antibodies; D003902:Detergents; D017396:Plasminogen Activator Inhibitor 2; D010450:Endopeptidases