FIGURE 3.

Catecholamines increase TfR1 mRNA stability and block Ft-H translation. A, effect of EPI and NE on TfR1 promoter activity. The mouse TfR1 promoter containing luciferase construct was transiently transfected to HepG2 cells along with CMV promoter containing β-galactosidase. After recovery, cells were kept untreated or treated with 30 μm EPI/NE. After 16 h, luciferase activity was measured in cell lysates and normalized with β-galactosidase activity. Cobalt chloride (100 μm) was used as a positive control. Results represent ± S.D. of three independent experiments performed in triplicates. B, effect of EPI/NE on TfR1 mRNA stability. HepG2 cells were treated with medium alone or EPI/NE (30 μm) for 6 h followed by 7.5 μg/ml actinomycin D treatment. Subsequently, total RNA was isolated after 0, 1, 2, and 3 h of actinomycin D treatment, and qRT-PCR was performed for TfR1 and actin. The figure represents data from three independent experiments. C, the upper panel shows the schematic diagram of the construct made in the pGL3-Control vector in which the 5′-UTR of Ft-H containing IRE was cloned upstream of luciferase gene. C2C12 cells were transfected with the same plasmid or only with pGL3-control vector along with β-galactosidase containing plasmid and then treated with EPI/NE (0–30 μm) for 16 h. Luciferase and β-galactosidase activities were measured in cell lysates and normalized (lower panels). Error bars represent ±S.D. from three independent experiments performed in triplicates.