Protective Effect of the HIF-1A Pro582Ser Polymorphism on Severe Diabetic Retinopathy

Figure 2

(a) HIF-1α (P402A/P564A/P582S) is as stable as HIF-1α (P402A/P564A). Human embryonic kidney 293A (HEK293A) cells were transiently transfected with hypoxia-inducible factor-1α (HIF-1α) (P402A/P564A) or HIF-1α (P402A/P564A/P582S). They were maintained in hypoxia (1% O₂) for 16 h before harvest. HIF-1α (P402A/P564A) and HIF-1α (P402A/P564A/P582S) were detected using a FLAG antibody. The HIF-1 constructs are equally expressed. α-Tubulin is shown as internal control. PPA, HIF-1α (P402A/P564A); HIF-1α-PPAPS, hypoxia-inducible factor-1α (P402A/P564A/P582S). (b) HIF-1α (P402A/P564A/P582S) is destabilized in hyperglycemic hypoxic conditions. Human embryonic kidney 293A (HEK293A) cells were transiently transfected with hypoxia-inducible factor-1α (HIF-1α) (P402A/P564A/P582S). They were exposed to 5.5 mM or 30 mM glucose and maintained in normoxia (N) (21% O₂) or hypoxia (H) (1% O₂) for 6 h before harvest. HIF-1α (P402A/P564A/P582S) was detected using a FLAG antibody. HIF-1α (P402A/P564A/P582S) was destabilized in hyperglycemic hypoxic conditions. HIF-1α-PPAPS, hypoxia-inducible factor-1α (P402A/P564A/P582S); N, normoxia; H, hypoxia. (c) HIF-1α (P402A/P564A/P582S) had a higher transactivation activity than HIF-1α (P402A/P564A). Human embryonic kidney 293A (HEK293A) cells were transiently transfected with hypoxia-inducible factor-1α (HIF-1α) (P402A/P564A/P582S) or HIF-1α (P402A/P564A) together with reporter plasmids in a dual-luciferase reporter assay. The cells were cultured in media containing 5.5, 20, and 30 mM of glucose for 48 h and maintained in hypoxia (1% O₂) for 6 h before harvest. PPA, HIF-1α (P402A/P564A); PPAPS, HIF-1α (P402A/P564A/P582S). The transactivation activity was significantly increased in HEK293A cells transfected with HIF-1α (P402A/P564A/P582S) compared to HIF-1α (P402A/P564A) (). Data are expressed as after two-way ANOVA with repeated measures, per group.

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