Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.     

Reduced growth hormone receptor messenger ribonucleic acid in an aged man with chronic malnutrition and growth hormone resistance.

A severely malnourished 87-yr-old man presented with hypoglycemia. Serum GH levels were elevated, and serum levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3, and GH-binding protein were extremely reduced. The patient's GH was biologically active. Administration of GH for 4 consecutive days resulted in a slight increment in serum IGF-I levels, but no elevation of serum IGF-binding protein-3. The expression of GH receptor messenger ribonucleic acid in the liver was greatly reduced. An autopsy revealed a Rathke's cleft cyst confined to the sella turcica. Immunohistochemical studies for GH showed that there was nothing to suggest a tumor overproducing GH. In addition, TSH levels were elevated in the presence of normal thyroid hormone levels, and there was a cluster of cells showing strong immunohistochemical staining for the TSH beta-subunit in the pituitary. In this patient, the decreased expression of GH receptor messenger ribonucleic acid in the liver may have been responsible for the GH resistance, which was probably caused by malnutrition.     

Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger ribonucleic acid

The testis is rich in central nervous system-type neuropeptides, including a GH-releasing hormone (GHRH)-like substance. We examined the ontogeny and pituitary regulation of testicular GHRH-like mRNA (t-GHRH mRNA) and compared this to expression of insulin-like growth factor-I (IGF-I) and IGF-II mRNA in developing testis. t-GHRH mRNA was measured by dot blot hybridization and quantitated using a hypothalamic GHRH cRNA standard. t-GHRH mRNA was not detectable in Northern blots in fetal testis on day 19 of gestation, but was present in low but detectable amounts in testicular dot blots on day 2 of life (0.44 pg/micrograms total RNA). Levels of the RNA increased beginning on day 21 (1.72 +/- 0.23 pg/micrograms total RNA) and reached adult levels by day 30 (4.96 +/- 0.84 pg/micrograms total RNA). The GHRH species on Northern analysis was about 1750 nucleotides at all ages examined; there was a larger species of about 3350 nucleotides seen on days 65 and 90. There was no correlation between the ontogeny of t-GHRH mRNA and either IGF-I or IGF-II mRNAs, which were maximally expressed in the testes of day 2 animals and decreased with age. To examine the influence of the pituitary gland on t-GHRH mRNA, levels of the mRNA were measured in the tests of hypophysectomized animals and age-matched controls. In animals hypophysectomized on day 21 and killed on day 42 and in animals hypophysectomized on day 42 and killed on day 63, there was marked diminution of t-GHRH mRNA (19 +/- 5% and 9 +/- 2% of age-matched controls, respectively). In contrast, in animals hypophysectomized on day 65 and killed on either day 80 or 90, there was a much smaller difference in levels of t-GHRH mRNA compared to values in control animals (73 +/- 20%). This was unlike the effect of hypophysectomy on testicular IGF-I mRNA, where uniform diminution was seen in all three groups. Because GH is important in the regulation of hypothalamic GHRH mRNA, we examined the effects of administration of recombinant human GH on the reinduction of t-GHRH mRNA after hypophysectomy and compared this to the reinduction of IGF-I mRNA. Neither t-GHRH mRNA nor testicular IGF-I mRNA increased in hypophysectomized animals treated with GH. Our results indicate that t-GHRH mRNA is developmentally regulated, and that the hypothalamic-pituitary axis is important in its expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

An antisense oligodeoxynucleotide to growth hormone messenger ribonucleic acid inhibits lymphocyte proliferation

The role of GH in lymphocyte proliferation was studied by examining the effect of an antisense oligodeoxynucleotide (ODN) complementary to GH mRNA. The results of these studies showed that antisense GH ODN treatment inhibits lymphocyte production of immunoreactive GH (irGH). Lymphocytes treated with the GH antisense ODN produced less irGH than did lymphocytes treated with control sense GH ODN. Antisense GH ODN-mediated inhibition of irGH production resulted in a decrease in lymphocyte proliferation. Cells with the antisense GH ODN had less (87%) incorporation of [3H]thymidine [( 3H]TdR) in both resting and Concanavalin-A-stimulated lymphocytes, whereas the incorporation of [3H]TdR in cells treated with a control ODN was not significantly affected. The effect of the antisense ODN on [3H]TdR incorporation was specific, since it could be reversed by hybridization competition with a complementary GH sense ODN or by the addition of exogenous rat GH. Collectively, the data indicate that lymphocytes synthesize and secrete irGH and that irGH produced by these cells can stimulate proliferation, suggesting that GH may play an autocrine/paracrine role in lymphocyte replication.     

The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway.

Evidence has accumulated indicating that LHRH might behave as an autocrine/paracrine growth inhibitory factor in some peripheral tumors. However, LHRH receptors in tumor cells have not been fully characterized, so far. The present experiments were performed to analyze: 1) the messenger RNA expression; 2) the molecular size; and 3) the signal transduction pathway of LHRH receptors in prostate cancer. For these studies, the human androgen-dependent LNCaP and androgen-independent DU 145 prostate cancer cell lines were used. 1) By RT-PCR, a complementary DNA product, which hybridized with a 32P-labeled oligonucleotide probe specific for the pituitary LHRH receptor complementary DNA, was found both in LNCaP and in DU 145 cells. 2) Western blot analysis, using a monoclonal antibody raised against the human pituitary LHRH receptor, revealed the presence of a protein band of approximately 64 kDa (corresponding to the molecular mass of the pituitary receptor) in both cell lines. 3) In LNCaP and DU 145 cells, pertussis toxin completely abrogated the antiproliferative action of a LHRH agonist (LHRH-A). Moreover, LHRH-A substantially antagonized the pertussis toxin-catalyzed ADP-ribosylation of a Galpha(i) protein. Finally, LHRH-A significantly counteracted the forskolin-induced increase of intracellular cAMP levels in both cell lines. These data demonstrate that the LHRH receptor, which is present in prostate cancer cells, independently of whether they are androgen-dependent or not, corresponds to the pituitary receptor, in terms of messenger RNA expression and protein molecular size. However, at variance with the receptor of the gonadotrophs, prostate cancer LHRH receptor seems to be coupled to the Galpha(i) protein-cAMP signal transduction pathway, rather than to the Galpha(q/11)-phospholipase C signaling system. This might be responsible for the different actions of LHRH in anterior pituitary and in prostate cancer.     

Thyroid hormone and growth hormone interact to regulate insulin-like growth factor-I messenger ribonucleic acid and circulating levels in the rat

Thyroid hormones influence growth in part by altering the secretion and effects of GH. GH, in turn, mediates its effects by regulating the synthesis and secretion of insulin-like growth factor-I (IGF-I). IGF-I is a pleiotropic growth factor that is synthesized by many tissues and acts on many tissues to regulate both cellular replication and differentiated function. We have studied the direct effects of thyroid hormones and the combined effects of thyroid hormones and GH on the regulation of IGF-I synthesis and secretion in hypophysectomized (hypox) rats in vivo. All rats, except normal littermates and a hypox control group, received 100 micrograms hydrocortisone/100 g BW for 10 days. Circulating IGF-I was measured by specific RIA (normal rats, 1 U/ml), and hepatic IGF-I mRNA was measured by Northern blot hybridization with an antisense cRNA probe. 1) Hypox rats treated with hGH (75 micrograms, ip, twice daily) for 10 days gained 17 g BW vs. 70 g for normal littermates. GH markedly increased hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 U/ml 12 h after the last GH injection vs. 0.03 +/- 0.02 for hypox controls). 2) T4 (1 micrograms/100 g BW, ip) for 10 days increased neither weight, hepatic IGF-I mRNA, nor circulating IGF-I. 3) Rats treated with T4 for 10 days followed by a single injection of 1 mg GH, ip, increased hepatic IGF-I mRNA and circulating IGF-I levels comparably as in rats receiving acute GH alone (IGF-I, 12 h, 0.31 +/- 0.09 vs. 0.36 +/- 0.06 U/ml). 4) Hypox rats treated with a single injection of T3 (1.5 micrograms/100 g BW, ip) had slightly increased hepatic IGF-I mRNA, but showed no significant change in circulating IGF-I levels. 5) A single injection of T3 plus GH to hypox rats increased IGF-I mRNA levels above those in rats injected with GH alone and increased serum IGF-I levels to 0.48 +/- 0.12 U/ml compared to 0.36 +/- 0.06 U/ml for GH alone. 6) After 10 days of GH treatment, a single injection of T3 lowered both hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 to 0.16 +/- 0.06 U/ml, 6 h after T3). These studies demonstrate that thyroid hormones have relatively little direct effect on IGF-I synthesis but can have major effects on GH-stimulated IGF-I synthesis and secretion. The pattern of these effects depends on the integrity of the pituitary gland, prior exposure of the liver to GH and/or thyroid hormones, and the temporal relationship between GH and thyroid hormone administration.