Hydrocortisone Regulates the Dynamics of Plasminogen Activator and Plasminogen Activator Inhibitor Expression in Cultured Murine Keratinocytes

Abstract

The plasminogen activators tPA and uPA, and their inhibitors, PAI-1 and PAI-2, have been associated with epithelial homeostasis and wound healing. In these studies, we investigate the effect of the steroid hormone hydrocortisone, a commonly used therapeutic modality for skin, on PAs/PAIs in serum- and plasminogen-free primary cultures of murine keratinocytes. SDS–PAGE fibrin zymography showed that addition of 1 μM hydrocortisone to cultures significantly reduced tPA fibrinolytic activity in both cell extracts and conditioned medium. uPA activity in conditioned medium was similarly inhibited. Cells were also cultured in the presence of dibutyryl cyclic AMP (dbcAMP). dbcAMP (5 mM) alone enhanced uPA and tPA fibrinolytic activity in conditioned medium, but this increase was diminished in the presence of 1 μM hydrocortisone. Immunoblots revealed a three- to fivefold induction of free PAI-1 by hydrocortisone which was partially blocked by dbcAMP. Northern blots showed that PAI-1 mRNA increased threefold 2 h after addition of hydrocortisone and remained elevated at least 8 h. In contrast, uPA and tPA mRNA were unchanged over the same time course. uPA, tPA, and PAI-1 mRNA increased in the presence of dbcAMP; levels remained elevated at least 8 h. HC suppressed the induction of uPA and tPA by dbcAMP. Studies directed at identifying plasminogen mRNA showed that in this culture system, keratinocytes produce no plasminogen mRNA either in the presence or in the absence of hydrocortisone or dbcAMP. Collectively, these results show that keratinocyte plasminogen activator activity is suppressed by hydrocortisone as a function of increased PAI-1 combined with an attenuation of PA induction by agents that increase intracellular cAMP. These results provide additional information to further define the mechanism by which glucocorticoids inhibit wound healing.