Retinoic acids and thyroid hormone act synergistically with dexamethasone to increase growth hormone-releasing hormone receptor messenger ribonucleic acid expression

The effects of all-trans-retinoic acid (RA), 9-cis-retinoic acid (9cRA), and thyroid hormone (T3) on GH-releasing hormone receptor (GHRH-R) messenger RNA (mRNA) expression were studied using ribonuclease protection assay in the fetal rat pituitary gland and in MtT/S cells, a clonal GH cell line derived from an estrogen-induced somatotropic tumor in the rat. Although RA (1 microM), 9cRA (1 microM), or T3 (1 nM) alone showed little effect on GHRH-R mRNA expression in the MtT/S cells, each of these substances was found to act synergistically with dexamethasone (DEX; 500 nM) to increase GHRH-R mRNA expression. The effects of RAs and T3 were dose dependent, with maximum effects observed at 1 microM and 1 nM, respectively. The maximum effect of RAs or T3 was not further augmented by the addition of T3 or RAs, respectively. No apparent differences were observed in this study between the actions of RA and 9cRA. The Northern analyses showed that MtT/S cells express retinoic acid receptor alpha2 mRNA and thyroid hormone receptor beta2 mRNA, and DEX did not affect the levels of these mRNAs. This suggests that the role of DEX in enabling RAs or T3 to up-regulate GHRH-R mRNA levels is not an induction of the expression of each specific receptor for RAs and T3. The similar enhancement of DEX induction of GHRH-R mRNA by RAs or T3 was also observed in the fetal rat pituitary gland in culture, suggesting that RA and/or T3 is involved in the mechanisms responsible for the developmentally regulated expression of GHRH-R mRNA.     

Regulation of retinoid and thyroid hormone action through homodimeric and heterodimeric receptors

Biologic responses to retinoids and thyroid hormones are mediated by their intracellular receptor proteins. Many exciting advances have been made recently in understanding the molecular mechanism by which these receptor proteins operate. In contrast to the steroid hormone receptors that function predominantly as homodimers, thyroid hormone receptors (TRs)and retinoic acid receptors (RARs) require interaction with the retinoid X receptors (RXRs) for efficient DNA binding and transactivation. In addition, RXRs, in the presence of their specific ligands such as 9-cis RA, can form homodimers that recognize a subset of retinoic acid responsive elements (RAREs). The retinoid responses mediated by RXR homodimers and RAR-RXR heterodimers can be restricted by the COUP-TF orphan receptors that bind strongly to certain RAREs as homodimers. Thus, a complex network of receptor interaction has been unraveled that promises a better understanding of thyroid and retinoid hormone regulation of fundamental biologic processes and diseases.     

Decreasing serum concentrations of all-trans, 13-cis retinoic acids and retinol during fasting and caloric restriction

OBJECTIVES: To investigate the effects of caloric restriction on the serum concentrations of retinoids in man. DESIGN: Samples were drawn before and during caloric restriction by fasting or 4-6 weeks after gastric surgery. SUBJECTS: The fasting group included 17 healthy subjects (11 women and six men) and 16 obese patients (10 women and six men) who underwent bariatric surgery (vertical banded gastroplasty). MAIN OUTCOME MEASURES: Serum concentrations of all-trans, 13-cis, 4-oxo-13-cis retinoic acids and retinol. RESULTS: The serum concentrations of retinol, all-trans and 13-cis retinoic acids decreased by about 20% after 5 days of fasting. After gastroplasty, the serum concentration of retinol, all-trans, 13-cis retinoic acids, retinol-binding protein and transthyretin also decreased to a similar extent after 1 month. In both groups we found a correlation between the delta values of 13-cis retinoic acid and its metabolite 4-oxo-13-cis retinoic acid. In all subjects there were also correlations between the delta values of the retinoids. However, these correlations were comparatively weak (e.g. r2 = 0.36 for retinol--all-trans retinoic acid). The change in retinoid concentrations did not correlate to the change of weight or body mass index. CONCLUSION: Our results support the hypothesis that serum retinol is one of the determinants of serum concentrations of all-trans and 13-cis retinoic acid and that the catabolism of 13-cis retinoic acid is not affected by fasting. However, in the individual case, S-Retinol is a poor predictor of S-All-trans retinoic acid.     

Multistep differentiation of GH-producing cells from their immature cells

In order to study GH cell differentiation, we used the clonal cell lines called MtT/E and MtT/S cells, which were derived from a rat mammotrophic pituitary tumor. Although MtT/E cells are non-hormone-producing ones, Pit-1 protein is present in their nuclei, which suggests that MtT/E cells are progenitor cells of the Pit-1 cell lineage and have the potential to differentiate into hormone-producing cells. On the other hand, MtT/S cells produce GH; however, the responsiveness to GH-releasing hormone (GHRH) is weak and only a small number of secretory granules are present in their cytoplasm, which suggests that MtT/S cells are premature GH cells. In order to differentiate into GH cells from MtT/E cells as a progenitor cell, we examined several differentiation factors and found that retinoic acid (RA) induced the differentiation of MtT/E cells into GH-producing cells. RA-induced GH cells partially matured with the glucocorticoid treatment; however, the responsiveness to GHRH on GH secretion was incomplete. In order to elucidate the mechanism underlying full differentiation of GH cells, we used MtT/S cells. We treated MtT/S cells with glucocorticoid and found that they differentiated into mature GH cells with many secretory granules in their cytoplasm and they responded well to GHRH. These results suggested that MtT/E and MtT/S cells are progenitor or premature GH cells, and show different responses to differentiation factors. Our data also suggested that GH cells differentiate from their progenitor cells through multistep processes.     

Stimulatory effect of retinoic acid on GH gene expression: the interaction of retinoic acid and tri-iodothyronine in rat pituitary cells

We have previously demonstrated that retinoic acid (RA) as well as thyroid hormone stimulates GH gene expression. To clarify the relationship between the action of RA and thyroid hormone, pituitary-specific gene expression was investigated further in rat pituitary cells. Rat clonal pituitary cells, GH3, were treated with RA with or without tri-iodothyronine (T3) for up to 3 days. After treatment with 10-1000 nmol RA/l with or without 0.1-10 nmol T3/l, medium was collected for radioimmunoassay and cells were subjected to RNA extraction, and GH and prolactin gene expression was analysed using 32P-labelled rat GH and rat prolactin cDNA probes respectively. The data demonstrated the dose-responsive manner of the stimulatory effects of RA and T3 on GH secretion with T3-depleted media. The action of RA was additive to that of T3 for GH secretion when maximum effective doses of RA or T3 were used. Using dot blot and Northern gel analysis, it was shown that RA increased GH mRNA levels in T3-depleted media, and that this action of RA was additive to that of T3 on the induction of GH mRNA levels. In contrast, neither RA nor T3 stimulated the secretion of prolactin and prolactin mRNA levels in these cells. Our results indicate that RA stimulates GH mRNA increment and GH secretion in T3-depleted media, and that the stimulatory effect of RA is additive to the maximum effective dose of T3.     

All-trans retinoic acid at low concentration directly stimulates normal adult megakaryocytopoiesis in the presence of thrombopoietin or combined cytokines.

In order to investigate the direct effects of retinoids on normal adult hematopoietic progenitors, purified CD34+ cells were seeded in serum-free cultures in the presence of pharmacological (10(-6)) M or physiological (10(-12)) M concentrations of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) plus combinations of specific cytokines. 10(-6) M ATRA and 9-cis RA significantly decreased the number of granulomacrophagic, erythroid and megakaryocytic (CFU-meg) progenitors. On the other hand, 10(-12) M ATRA significantly promoted the growth of CFU-meg, in the presence either of thrombopoietin or of IL-3+ GM-CSF, and induced a reproducible stimulation of the immature CD34+DR- subset. In conclusion, our findings suggest that retinoic acids probably play a direct role in normal adult hematopoietic development at both physiological and pharmacological concentrations. The stimulatory effect on megakaryocytopoiesis should be considered in the perspective of a potential use of low-dose ATRA, combined with thrombopoietin or other cytokines, in pathological conditions where the megakaryocytic compartment is impaired and the stimulation of megakaryocytopoiesis is requested.     

Retinoids and retinol differentially regulate steroid biosynthesis in ovarian theca cells isolated from normal cycling women and women with polycystic ovary syndrome

CONTEXT: Polycystic ovary syndrome (PCOS) is characterized by ovarian androgen excess and infertility. Recent experiments have suggested that several genes involved in retinoic acid synthesis may be differentially expressed in PCOS theca cells and may contribute to excessive theca-derived androgen production. OBJECTIVE: The study was performed to examine whether there are differential effects of retinol and retinoids on normal and PCOS theca cell function. DESIGN: We used in vitro assays. SETTING: The study was conducted at the university laboratory. PATIENTS: We studied theca interna cells isolated from normal-cycling women and women with PCOS. INTERVENTIONS: Theca cells were treated with all-trans-retinoic acid (atRA), 9-cis retinoic acid (9-cis RA), or the retinoic acid precursor retinol. MAIN OUTCOME MEASURE(S): We measured dehydroepiandrosterone, testosterone, and progesterone biosynthesis as well as cytochrome P450 17alpha-hydroxylase (CYP17), cytochrome P450 cholesterol side-chain cleavage, and steroidogenic acute regulatory protein mRNA abundance and promoter function. RESULTS: Dehydroepiandrosterone production was increased by atRA and 9-cis RA in normal cells and by atRA, 9-cis RA, and retinol in PCOS. Testosterone production was increased by atRA in normal and by atRA, 9-cis RA, and retinol in PCOS. Progesterone production was not altered by retinoid treatment. Retinoids stimulated mRNA abundance and promoter function for CYP17 and steroidogenic acute regulatory protein in both cell types and cytochrome P450 cholesterol side-chain cleavage in normal cells. Retinol stimulated CYP17 mRNA accumulation and promoter function in PCOS but not normal theca cells. P < 0.05 was considered statistically significant. CONCLUSIONS: Differential responses to retinol and retinoids in normal and PCOS theca suggest that altered retinoic acid synthesis and action may be involved in augmented CYP17 gene expression and androgen production in PCOS.     

Establishment of an estrogen responsive rat pituitary cell sub-line MtT/E-2.

A rat pituitary tumor sub-line MtT/E-2 was established from a rat pituitary tumor cell line MtT/E. Its growth was found to depend on the presence of estradiol (E2) in culture media at 10(-13)-10(-9) M, whereas the original cell line MtT/E proliferated autonomously. The recently discovered PTTG (pituitary tumor transforming gene) is highly expressed in this cell line, although not regulated by E2. On the other hand, E2 induced c-myc and cyclin D1 proteins in MtT/E-2, which contains a lot (220+/-18 fmol/mg protein) of estrogen receptor demonstrated by RT-PCR analysis to be predominantly alpha type. MtT/E-2 secretes growth hormone which is, interestingly, regulated by retinoic acid and dexamethasone rather than thyroid hormones.     

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone

GH is first detectable in the fetal rat pituitary between gestational days 18 and 19. The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear. We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function. In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day. These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid. The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures. GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats. The presence or absence of T3 in the culture medium did not alter the response to GHRH. In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course. Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses. IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions. The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted. Addition of T3 partially restored the responsiveness of these cells to IGF-I. The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary. Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance.     

Epidermal growth factor decreases thyroid hormone receptors and attenuates thyroid hormone responses in GH4C1 cells

The present study was undertaken to examine the effect of long term exposure to epidermal growth factor (EGF) on thyroid hormone responses as well as the concentration of specific nuclear thyroid hormone receptors in GH4C1 rat pituitary tumor cells. GH4C1 cells were first incubated for 48 h in medium with 5% fetal calf serum depleted of thyroid hormones by ion exchange resin. EGF had no effect on thyroid hormone receptors after 2 h, but decreased [125I]T3 binding to 56% of control values at 24 h and 68% at 48 h. L-T3 (0.5 nM) caused down-regulation of thyroid hormone receptors, and addition of EGF caused a further decrease. T3 alone (0.5 nM) caused a 2- to 3-fold induction of GH after 48 h, and GH induction was significantly inhibited by the addition of 10 nM EGF. Scatchard analysis of specific nuclear [125I]T3 binding showed that 48-h incubation with 10 nM EGF decreased T3 receptors from a Bmax of 2.35 to 1.26 pmol/mg DNA in thyroid hormone-depleted medium without affecting receptor affinity (Kd, 80 pM). The decrease in nuclear thyroid hormone receptors caused by EGF was dose dependent, with half-maximal inhibition at 0.10 nM EGF. EGF attenuated the GH response to T3 with similar dose-response characteristics. When cells were incubated for 48 h with different concentrations of T3, EGF (10 nM) decreased thyroid hormone receptors to 56-72% of control values regardless of the dose of T3, and EGF shifted the ED50 for T3 stimulation of GH from 0.1 to 1.2 nM. EGF also reduced from 5- to 1.8-fold the increase in cell number caused by thyroid hormone over 2 weeks. In contrast, EGF stimulation of PRL synthesis was changed only slightly by thyroid hormone at all times. In conclusion, we demonstrate that low concentrations of EGF decrease nuclear thyroid hormone receptors and thyroid hormone responses; this may be the mechanism by which EGF suppresses T3-induced GH production in GH4C1 cells.     

Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 cells

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.