Induction of PAI-1 mRNA and protein expression by insulin under both normoxia and mild hypoxia.
Cells were cultured for 24 hours under arterial pO2. At 24 hours the medium was changed, and cells were further cultured for the next 24 hours under normoxic (16% O2) and hypoxic (8% O2) conditions. Three hours before harvesting, the cells were treated with insulin, wortmannin, or both. The PAI-1 mRNA levels were measured by Northern blotting. (A) The PAI-1 mRNA level under normoxia (16% O2) without addition of insulin was set equal to 100%. Concentrations of insulin are indicated. (B) The PAI-1 mRNA level under normoxia (16% O2) without addition of insulin was set equal to 100%. Insulin was used at a concentration of 10 nM, wortmannin was used at a concentration of 20 nM. Values are expressed as means ± SEMs of 3 independent culture experiments. Student t test for paired values: *significant difference 16% O2 versus 8% O2, **significant difference 16% O2 + insulin versus 16% O2(control), ***significant difference 8% O2 + insulin versus 8% O2 (control), P ≤ .05. (C) Representative Northern and Western blot. For Northern analysis 15 μg total RNA was hybridized to digoxigenin-labeled PAI-1, GK, and β-actin antisense RNA probes (see “Materials and methods” for more information). Protein (50 μg) from the medium was subjected to Western analysis with an antibody against rat PAI-1 (see “Materials and methods” for more information). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry. Wo indicates wortmannin.
