1 2 COX-2 overexpression inhibits cathepsin D mediated cleavage of plasminogen to 3 the potent anti-angiogenic factor angiostatin 4 5 6 7 Gabrielle B Perchick and Henry N Jabbour 8 9 10 11 Abstract 1 Overexpression of COX-2 and enhanced synthesis of PGE 2 have been implicated in 2 human endometrial pathologies. To investigate the molecular role of COX-2, the 3 Ishikawa human endometrial epithelial cell line was stably transfected with the 4 pIRES2 vector containing COX-2 cDNA in either the

more »

... or antisense directions. 5 PGE 2 concentrations were significantly elevated in the cells transfected with the 6 COX-2 sense compared with wild type cells or cells transfected with the antisense 7 cDNA (P<0.01). Elevated PGE 2 synthesis was associated with enhanced expression 8 and signalling of EP receptors. cDNA array analysis revealed differential expression 9 of cathepsin D between the COX-2 sense and antisense cells. Cathepsin D RNA and 10 protein expression was 6.7 and 2.1 fold lower in the COX-2 sense compared with 11 COX-2 antisense cells respectively. Cathepsin D is known to cleave plasminogen to 12 the potent anti-angiogenic factor angiostatin. To investigate differential angiostatin 13 generation, conditioned media from COX-2 sense, COX-2 antisense and wild type 14 cells were incubated with plasminogen and subsequently subjected to Western blot 15 analysis. In comparison to wild type cells, the cleavage of plasminogen to angiostatin 16 was abolished when incubated in COX-2 sense cells conditioned media and elevated 17 when incubated in COX-2 antisense cells conditioned media. Co-incubation of 18 plasminogen with the cathepsin D inhibitor Pepstatin A inhibited the cleavage of 19 plasminogen to angiostatin in the COX-2 antisense conditioned media. These data 20 demonstrate that COX-2 exerts a negative feedback on the expression of cathepsin D. 21 This in turn reduces the generation of the anti-angiogenic factor angiostatin, hence 22 promoting a pro-angiogenic environment. 23 24 Word count = 249 25