Thyroxine increases the levels of epidermal growth factor messenger ribonucleic acid (EGF mRNA) in the thyroid in vivo, as revealed by quantitative reverse transcription polymerase chain reaction with an internal control EGF mRNA

Abstract

Treating mice with T4 increases the level of immunoreactive epidermal growth factor (EGF) in the thyroid. In order to establish whether this response might reflect a T4-dependent increase in the levels of messenger RNA (mRNA) in the thyroid, we prepared an internal standard which permits us to quantitate EGF mRNA levels by reverse transcription plus polymerase chain reaction amplification (RT-PCR). Our synthetic EGF mRNA construct contains the same flanking primer sequences used to amplify mature EGF message, but 70 bases were eliminated from the center of the 277-base EGF sequence to permit the PCR product of this internal standard to be distinguished by its smaller size (EGF 207). This synthetic mRNA also contains a poly(A)tail, which permits it to be reverse transcribed. We then added a range of concentrations of this internal standard mRNA to aliquots of total RNA from each pair of thyroid lobes and determined the concentration of EGF 207 at which the PCR primers were incorporated equally into the 277 and 207 bands after RT-PCR. Thyroid RNA from male Balb/c mice treated with T4 (0.25 micrograms/g.day) for 14 days contained 2.8-fold more EGF mRNA than RNA from control mice (P < 0.01). Competitive RT-PCR EGF mRNA levels were determined for thyroid RNA samples from mice treated with T4 for various times up to 14 days. The most significant increase occurred after 1 day's treatment (P < 0.005). This demonstration of a thyroid hormone-dependent increase in the level of thyroidal EGF mRNA adds support to the concept that EGF may function as an autocrine/paracrine regulator of thyroid function.