Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.     

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.     

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.     

Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic peptide inhibits iodide uptake and thyroglobulin messenger ribonucleic acid expression in cultured bovine thyroid follicles

The involvement of atrial natriuretic peptide (ANP) in the regulation of thyroid gland is supported by the presence of high-affinity ANP receptors and the identification of the peptide in thyroid follicular cells. The aim of this work was to study the action of ANP on parameters of thyroid hormone biosynthesis and analyze the intracellular mechanism of the ANP action in cultured bovine thyroid follicles. The addition of ANP (0.1-10 nM) to the culture medium for 24 h inhibited the TSH (thyroid-stimulating hormone)-stimulated iodide uptake with a maximal inhibition at 1 nM ANP. When thyrocytes were incubated with 10 nM ANP the inhibitory effect slightly increased from 24 to 72 h. Thyroglobulin (Tg) mRNA expression was reduced by 1 and 10 nM ANP. After 24 h of treatment with the cGMP analogue, N(2),2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate [(Bu)(2)cGMP] (0.1 and 1 mM), an inhibition of iodide uptake and Tg mRNA expression was obtained, evidencing a cGMP-mediated inhibitory signal in the thyroid cell. A reduction of the cAMP production was induced by incubation of thyroid follicles with 1 and 10 nM ANP for 24 h. Under a similar treatment the cGMP accumulation was increased only by 10 nM ANP. The inhibitory effect of ANP on Tg mRNA level was reverted in the presence of pertussis toxin, an inhibitor of the G(i)-protein-mediated reduction of the adenylate cyclase activity. These results indicate an inhibitory action of ANP on parameters of thyroid hormone biosynthesis. A G(i)-protein-mediated reduction of the cAMP production seems to be the main factor involved in the ANP action although a role of the cGMP pathway should not be discarded specially at high ANP levels.     

Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells

We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c-fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c-myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP.     

Estrogen effects on histone messenger ribonucleic acid levels in the rat uterus

RNA was isolated from uteri of immature rats before and after estrogen treatment. The concentration of histone mRNA was analyzed by Northern hybridization and compared with messenger RNA concentration of alpha-actin, beta-actin, and beta-tubulin. Steady state levels of common histone mRNAs did not change up to 9 h after hormone administration. After that time the histone mRNA levels increased significantly and reached a maximum at 18 h, several hours later than the time of maximal histone protein biosynthesis induced by estrogen. The concentration of control mRNAs (alpha- and beta-actin and beta-tubulins) increased shortly after estradiol injection and reached a peak at 9 h. These results show that the pattern of histone gene expression induced by estrogen has some features similar to those observed during embryogenesis.     

Thyrotropin stimulation of lysosomal tyrosine transport in rat FRTL-5 thyroid cells

Tyrosine countertransport was used to demonstrate the hormonal stimulation of neutral amino acid transport across the lysosomal membrane of FRTL-5 cells. Cells grown with thyrotropin (1 X 10(-10) M) had 7-fold (+/- S.E.) higher tyrosine countertransport activity in their lysosomes than cells grown without thyrotropin. Thyrotropin also stimulated the uptake into tyrosine-loaded lysosomes of other neutral amino acids recognized by the tyrosine carrier, namely, phenylalanine (3-fold) and leucine (6-fold). In contrast lysosomal cystine countertransport was not affected by thyrotropin. Addition of thyrotropin to cells grown without thyrotropin showed that the stimulation of tyrosine counter-transport (a) required at least 48 h to reach the level of the thyrotropin-supplemented cells, (b) depended upon protein synthesis, since cycloheximide (20 microM) was inhibitory, and (c) depended upon RNA synthesis, since actinomycin D (1 nM) was inhibitory. Cells grown without thyrotropin but with dibutyryl cyclic AMP (1 mM) or cholera toxin (1 nM) exhibited enhanced lysosomal countertransport of tyrosine, suggesting that cyclic AMP may act as a messenger. This represents the first demonstration of hormonal responsiveness in a lysosomal transport system and may reflect the importance of salvage and reutilization of lysosomal degradation products for the thyroid epithelial cell.