Cyclooxygenase-2 overexpression inhibits cathepsin D-mediated cleavage of plasminogen to the potent antiangiogenic factor angiostatin.

Overexpression of cyclooxygenase (COX)-2 and enhanced synthesis of prostaglandin E2 (PGE2) have been implicated in human endometrial pathologies. To investigate the molecular role of COX-2, the Ishikawa human endometrial epithelial cell line was stably transfected with the pIRES2 vector containing COX-2 cDNA in either the sense or antisense directions. PGE2 concentrations were significantly elevated in the cells transfected with the COX-2 sense compared with wild-type cells or cells transfected with the antisense cDNA (P < 0.01). Elevated PGE2 synthesis was associated with enhanced expression and signaling of PGE2 receptors (EP). cDNA array analysis revealed differential expression of cathepsin D between the COX-2 sense and antisense cells. Cathepsin D RNA and protein expression was 6.7- and 2.1-fold lower in the COX-2 sense compared with COX-2 antisense cells respectively. Cathepsin D is known to cleave plasminogen to the potent antiangiogenic factor angiostatin. To investigate differential angiostatin generation, conditioned media from COX-2 sense, COX-2 antisense and wild-type cells were incubated with plasminogen and subsequently subjected to Western blot analysis. In comparison to wild-type cells, the cleavage of plasminogen to angiostatin was abolished when incubated in COX-2 sense cells conditioned media and elevated when incubated in COX-2 antisense cells conditioned media. Coincubation of plasminogen with the cathepsin D inhibitor pepstatin A inhibited the cleavage of plasminogen to angiostatin in the COX-2 antisense conditioned media. These data demonstrate that COX-2 exerts a negative feedback on the expression of cathepsin D. This in turn reduces the generation of the antiangiogenic factor angiostatin, hence promoting a proangiogenic environment.