THYROXINE INCREASES THE LEVELS OF EPIDERMAL GROWTH-FACTOR MESSENGER-RIBONUCLEIC-ACID (EGF MESSENGER-RNA) IN THE THYROID INVIVO, AS REVEALEDBY QUANTITATIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION WITH AN INTERNAL CONTROL EGF MESSENGER-RNA

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 messe nger RNA (mRNA) in the thyroid, we prepared an internal standard which permits us to quantitate EGF mRNA levels by reverse transcription plu s polymerase chain reaction amplification (RT-PCR). Our synthetic EGF mRNA construct contains the same flanking primer sequences used to amp lify mature EGF message, but 70 bases were eliminated from the center of the 277-base EGF sequence to permit the PCR product of this interna l standard to be distinguished by its smaller size (EGF 207). This syn thetic mRNA also contains a poly(A)tail, which permits it to be revers e transcribed. We then added a range of concentrations of this interna l standard mRNA to aliquots of total RNA from each pair of thyroid lob es and determined the concentration of EGF 207 at which the PCR primer s were incorporated equally into the 277 and 207 bands after RT-PCR. T hyroid RNA from male Balb/c mice treated with T4 (0.25 mug/g . day) fo r 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 thy roid 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 E GF may function as an autocrine/paracrine regulator of thyroid functio n.