Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Abstract

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+-specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4, 0.4 μg/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 ± 0.9%, mean ± SEM) than in that of control mice (7.3 ± 0.5%, P < 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (Mrs) of 12 000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with α-chymotrypsin. Autoradiograms, following high-voltage electrophoresis and ascending chromatography on thin-layer cellulose, showed a marked similarity of the peptide maps of purified EGF, the IMMP of a SMG extract, and the translation product IMMPs. Furthermore, the peptide maps of the Mr 12 000, 9000, and 6000 IMMPs were highly concordant, suggesting that the Mr 12 000 and 9000 species are structurally related to the Mr 6000 EGF species. These observations support the hypothesis that thyroid hormones increase SMG EGF concentrations by increasing the number of poly(A)+ RNA species coding for EGF.