ADA-complexing protein CD26 is coordinately induced by hypoxia. (A) Real-time PCR was used to confirm induction of CD26 mRNA by hypoxia in cultured endothelial cells (HMEC-1s and HUVECs). Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over normoxia ± SD at each indicated time. Results are derived from 3 experiments in each condition (^*P < .01). (B) Increase in total CD26 protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia, washed, and lysed. Lysates were resolved by SDS-PAGE, and resultant Western blots were probed with mAb directed against human CD26. A representative experiment of 3 is shown. (C) Increase in surface CD26 protein with hypoxic exposure. Confluent HMEC-1 monolayers were exposed to indicated periods of hypoxia, monolayers were washed, surface proteins were biotinylated, and cells were lysed. CD26 was immunoprecipitated with mAb directed against human CD26. Immunoprecipitates were resolved by SDS-PAGE, and resultant Western blots were probed with avidin-peroxidase. A representative experiment of 3 is shown. (D) ADA surface induction requires interaction with CD26. To confirm that the observed increase in enzymatically active cell-surface ADA is bound to hypoxia induced CD26, HIV-1 gp120, a specific inhibitor of ADA interaction with CD26, was used. Postnormoxic or posthypoxic (pO₂ 20 mm Hg, 48 hours) HMEC-1s were incubated for 10 minutes with 100 nM gp120 in HBSS at 37°C and washed; ADA activity was measured. Note the reduced ADA activity after gp120 treatment in postnormoxic and posthypoxic endothelia (P < .01 by ANOVA). (A, D) Error bars indicate SD.