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Urea, in concentrations unique to the renal medulla, increases transcription and protein expression of several immediate-early genes (IEGs) including the zinc finger-containing transcription factor, Egr-1. In the present study, the proximal 1.2 kb of the murine Egr-1 5' -flanking sequence conferred urea-responsiveness to a heterologous luciferase reporter gene when transiently transfected into renal medullary mIMCD3 cells,and this effect was comparable with that of the extremely potent immediate-early gene inducer, O-tetradecanoylphorbol 13-acetate (TPA). Urea inducibility of Egr-1 expression was protein kinase C (PKC)-dependent because staurosporine and calphostin C abrogated the urea effect, and down-regulation of PHC through chronic TPa treatment inhibited both urea-inducible Egr-1 protein expression and gene transcription. In addition, hyperosmotic urea increased inositol 1,4,5-trisphosphate (IP3) release from mIMCD3 cells and induced tyrosine phosphorylation of the receptor tyrosine kinase-specific phospholipase C (PLC) isoform, PLC-gamma. Importantly, urea-inducible Egr-1 expression was strongly genistein-sensitive, to a much greater extent than the comparable TPA-inducible Egr-1 expression. These data suggest that urea-inducible Egr-1 expression is a consequence of sequential PLC-gamma activation, IP3 release, and PKC activation. Urea-inducible PLC-gamma activation, in conjunction with the genistein-sensitivity of urea-inducible Egr-1 expression suggest the possibility of a cell surface or cytoplasmic urea-sensing receptor tyrosine kinase.

Follistatin is a glycoprotein, originally isolated from the gonads, that specifically inhibits follicle-stimulating hormone (FSH) biosynthesis and secretion. We have previously detected follistatin mRNA in rat pituitary gonadotropes. To assess the potential physiologic role of follistatin in the rat pituitary, we have investigated the effects of gonadectomy (GDX) and of sex steroid replacement on pituitary follistatin gene expression. Follistatin mRNA levels in individual rat pituitaries were measured by a quantitative reverse transcription-polymerase chain reaction assay. Female and male rats 21 d old underwent surgical GDX and were then killed 21 d after GDX. Follistatin mRNA levels in rat pituitary increased 3.2 +/- 1.5-fold (P < 0.01) in GDX female rats and 8.2 +/- 2.0-fold (P < 0.005) in GDX male rats, compared with intact female and male controls, respectively. Replacement therapy with 17 beta-estradiol-3-benzoate (10 micrograms/100 g body weight) subcutaneously daily for 7 d in GDX female rats resulted in a slight further increase in follistatin mRNA levels compared to GDX females. In contrast, therapy with testosterone propionate (500 micrograms/100 g body weight) subcutaneously daily for 7 d in GDX male rats resulted in a decrease in follistatin mRNA levels, towards but not completely back to baseline levels in intact males. Time-course studies in adult male and female rats showed that the increase in follistatin mRNA levels after GDX is rapid, with significant increases occurring within 24 h after GDX, and parallels or precedes increases in FSH beta mRNA levels and FSH secretion. The regulation of follistatin mRNA levels in the rat pituitary by GDX and by sex steroids suggests that follistatin may be important as an autocrine or paracrine factor in the regulation of FSH.

Thyroid hormone (T3) has been shown to regulate the level of its receptor in a number of tissues and cell lines. Recently, proteins encoded by the protooncogene c-erbA have been identified as T3 receptors. In the rat, four c-erbA gene products have been isolated, three of which, r-erbA alpha-1, r-erbA beta-1, and r-erbA beta-2, encode biologically active T3 receptors; the fourth, r-erbA alpha-2, may play an inhibitory role in T3 action. The present work examines the molecular nature of T3 receptor autoregulation using probes specific for each c-erbA mRNA. Rats were rendered hypothyroid with propylthiouracil and then treated with either saline or T3. Northern blot analyses reveal marked tissue-specific and differential regulation of the multiple c-erbA mRNAs by T3. In the pituitary the levels of r-erbA beta-1 mRNA increase, whereas the levels of the pituitary-specific r-erbA beta-2 mRNA decrease with T3 treatment. In heart, kidney, liver, and brain the levels of r-erbA beta-1 are unaffected by thyroidal status. The levels of both r-erbA alpha mRNAs decrease with T3 treatment in all tissues examined except for the brain, where there is no change. In addition, we find that changes in the mRNAs encoding specific subpopulations of T3 receptors do not always parallel changes in total nuclear T3 binding. Differential regulation of the specific c-erbA mRNA species could have important consequences for T3 action.

Follicle-stimulating hormone (FSH) beta, luteinizing hormone (LH) beta, and alpha subunit messenger RNA (mRNA) levels were examined in rats after castration and sex-steroid replacement. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta complementary DNA (cDNA). Rat FSH beta mRNA is 1.7 kilobase in size. After ovariectomy, female FSH beta mRNA levels increased fourfold, whereas those of LH beta and alpha increased twenty- and eightfold, respectively. With estradiol, all subunits returned toward normal levels. Male LH beta and alpha mRNA levels rose eight- and fourfold, respectively, 40 d postcastration, but FSH beta mRNA levels increased minimally. After 7 d of testosterone propionate, LH beta and alpha mRNAs declined to normal levels, whereas FSH beta mRNA increased slightly. We conclude that in female rats FSH beta is negatively regulated by gonadal steroids, but to a lesser extent than LH beta or alpha mRNAs, and there is a differential regulation of FSH beta mRNA levels in males as compared with females at the time points examined.

Gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Previously we have reported the cloning of the messenger RNAs (mRNAs) and gene that encode human GRP. We now report that GRP mRNAs are markedly elevated in human fetal lung during the canalicular phase of pulmonary development (from approximately 16 to 30 wk gestation). By RNA blot and in situ hybridization analyses, GRP mRNAs were first detectable in fetal lung at 9-10 wk, plateaued at levels 25-fold higher than in adult lungs from 16 to approximately 30 wk and then declined to near adult levels by 34 wk gestation. By contrast, GRP peptide levels remain elevated until several months after birth. Consistent with this, in situ hybridization and immunohistochemical studies showed that GRP mRNA and peptide consistently colocalized in early gestation lung but that in neonatal lung, many cells that contained GRP peptide no longer contained GRP mRNA. The transient expression of high levels of GRP mRNAs during an approximately 12-wk phase of fetal lung development suggests that the secretion of GRP or its COOH-terminal peptides from pulmonary neuroendocrine cells may play a role in normal lung development.

Little is known about the hormonal regulation of luteinizing hormone (LH) biosynthesis. We have studied the regulation of LH messenger RNA (mRNA) levels by gonadal-steroid hormones in the rat. In one set of experiments, male and female rats were surgically gonadectomized (GDX) and killed 1, 3, 7, 14, 22, and 31 d postoperatively. In another set of experiments, male and female rats were surgically GDX and were injected subcutaneously with testosterone propionate (500 micrograms/100 g body wt per d) or 17 beta-estradiol 3-benzoate (10 micrograms/100 g body wt per d), respectively, beginning 3 wk postoperatively. Levels of serum LH were determined by radioimmunoassay and levels of LH subunit mRNAs in single pituitary glands were determined by blot hybridization analysis using labeled synthetic oligodeoxyribonucleotide probes that correspond to portions of the coding regions of the rat alpha- and LH beta-subunit mRNAs. 4 wk after gonadectomy, serum LH levels rose nine- and 20-fold, while alpha-subunit mRNA levels rose six- and 10-fold, and LH beta-subunit levels rose seven- and 14-fold, compared with controls in males and females, respectively. In gonadal-steroid hormone-treated male and female GDX rats, serum LH levels fell to 8 and 36% of control values, while alpha-subunit mRNA levels declined to 22 and 19%, and LH beta-subunit mRNA levels declined to 6 and 10% of control values, 48 h after injections were initiated, in males and females, respectively. We conclude that gonadal-steroid hormones negatively regulate the levels of both subunit mRNAs in GDX rats in a pattern that parallels the changes in serum LH values. These data suggest that gonadal-steroid hormone regulation of LH biosynthesis occurs, at least in part, at the level of LH subunit mRNAs due to effects at the transcriptional and/or RNA stability levels.