GHRH proliferative action on somatotrophs is cell-type specific and dependent on Pit-1/GHF-1 expression.

To investigate the mechanisms by which the hypothalamic peptide GHRH influences cell division, we analyzed its effects on the proliferation of two different cell lines: CHO-4, an ovary-derived cell line, and GH3, a pituitary-derived cell line. We found that GHRH induces the proliferation of pituitary-derived cells but inhibits the proliferation of ovary-derived cells. We further characterized this dual effect of GHRH to find that the cytoplasmic signals induced by this hormone are similar in both cell lines. Moreover, in CHO-4 cells GHRH stimulates two well-known positive cell cycle regulators, c-myc and cyclin D1, but is unable to induce the degradation of the negative cell cycle regulator p27(Kip1). Significantly, when the Pit-1/GHF-1 gene is exogenously expressed in CHO-4 cells, the negative effect of GHRH on the proliferation of these cells is attenuated. Furthermore, when the levels of Pit-1 are downregulated by siRNA in GH3-GHRHR cells, the positive effects of GHRH on the proliferation of these cells are diminished. These findings add to our understanding of the molecules involved in the regulation of cell proliferation by GHRH, as we demonstrate for the first time that Pit-1 is not only required to drive the expression of the GHRH receptor, as previously described, but is also needed for the downstream effects that occur after its activation to modulate cell proliferation. These data suggest that the regulation of cell proliferation in response to a specific growth factor depends in certain cell populations on the presence of a tissue-specific transcription factor.

Rewiring of the apoptotic TGF-ß-SMAD/NFκB pathway through an oncogenic function of p27 in human papillary thyroid cancer.

Papillary thyroid carcinoma (PTC), the most frequent thyroid cancer, is characterized by low proliferation but no apoptosis, presenting frequent lymph-node metastasis. Papillary thyroid carcinoma overexpress transforming growth factor-beta (TGF-ß). In human cells, TGF-ß has two opposing actions: antitumoral through pro-apoptotic and cytostatic activities, and pro-tumoral promoting growth and metastasis. The switch converting TGF-ß from a tumor-suppressor to tumor-promoter has not been identified. In the current study, we have quantified a parallel upregulation of TGF-ß and nuclear p27, a CDK2 inhibitor, in samples from PTC. We established primary cultures from follicular epithelium in human homeostatic conditions (h7H medium). TGF-ß-dependent cytostasis occurred in normal and cancer cells through p15/CDKN2B induction. However, TGF-ß induced apoptosis in normal and benign but not in carcinoma cultures. In normal thyroid cells, TGF-ß/SMAD repressed the p27/CDKN1B gene, activating CDK2-dependent SMAD3 phosphorylation to induce p50 NFκB-dependent BAX upregulation and apoptosis. In thyroid cancer cells, oncogene activation prevented TGF-ß/SMAD-dependent p27 repression, and CDK2/SMAD3 phosphorylation, leading to p65 NFκB upregulation which repressed BAX, induced cyclin D1 and promoted TGF-ß-dependent growth. In PTC samples from patients, upregulation of TGF-ß, p27, p65 and cyclin D1 mRNA were significantly correlated, while the expression of the isoform BAX-ß, exclusively transcribed in apoptotic cells, was negatively correlated. Additionally, combined ERK and p65 NFκB inhibitors reduced p27 expression and potentiated apoptosis in thyroid cancer cells while not affecting survival in normal thyroid cells. Our results therefore suggest that the oncoprotein p27 reorganizes the effects of TGF-ß in thyroid cancer, explaining the slow proliferation but lack of apoptosis and metastatic behavior of PTC.

Potent SHC tyrosine phosphorylation by epidermal growth factor at low receptor density or in the absence of receptor autophosphorylation sites.

The importance of epidermal growth factor (EGF) receptor expression level and autophosphorylation sites in src homology and collagen protein (SHC) tyrosine phosphorylation has been studied. In contrast to EGF-induced tyrosine phosphorylation of the GTPase-activating protein for ras (rasGAP) and phospholipase C-gamma 1 (PLC-gamma 1), SHC tyrosine phosphorylation occurs at a very low receptor density in parental NIH3T3 mouse fibroblasts expressing less than 1 x 10(4) EGF receptors per cell. In transfected NIH3T3 cells expressing human EGF receptors (approximately 4 x 10(5) receptors per cell), maximal levels of SHC and PLC-gamma 1 tyrosine phosphorylation occur when approximately 4 x 10(4) receptors or more are occupied by ligand. At lower levels of receptor occupancy only SHC phosphorylation was significant. Also, EGF treatment of mouse keratinocytes, which represent a physiological target of EGF, express a low number of EGF receptors (approximately 2 x 10(4) receptors per cell), and stringently require EGF to grow, results in intense SHC tyrosine phosphorylation, compared to rasGAP or PLC-gamma 1. SHC is also efficiently tyrosine phosphorylated by an EGF receptor deletion mutant (Dc214) that is devoid of autophosphorylation sites, but which remains mitogenically responsive to EGF. The EGF receptor mutant Dc214 is able to activate the ras guanine nucleotide exchanger and phosphorylate mitogen-activated protein kinase (MAPK), presumable as a result of complex formation between tyrosine phosphorylated SHC and GRB2. These results indicate that potent EGF-induced SHC tyrosine phosphorylation can be triggered in cells having relatively few receptors. Also, our data show that EGF receptors are able to phosphorylate SHC, activate the exchange of guanine nucleotide on ras and phosphorylate MAPK by a mechanism that does not require receptor autophosphorylation sites and, therefore, the src homology 2 (SH2):phosphotyrosine-dependent interaction of SHC or GRB2 with the EGF receptor.

TGF-beta1 actions on FRTL-5 cells provide a model for the physiological regulation of thyroid growth.

Little is known about the TGF-beta1 mechanism that promotes thyroid cell growth arrest. We assessed TGF-beta1 effects on Fisher rat thyroid cell line (FRTL-5). This allowed us to study TGF-beta1 action on thyroid cells in various physiological situations such as actively proliferating cells, resting cells stimulated to proliferate by the action of various mitogens, and resting cells. TGF-beta1 arrested proliferating FRTL-5 cells, increasing c-myc mRNA levels and reducing p27-free cyclin D1 protein levels, without affecting either the cellular content of p27 or the cyclin D1-p27 complexes. Moreover, TGF-beta1 treatment reduced the activity of cyclin E-CDK2 complexes and, consequently, pRB was found to be hypophosphorylated. TGF-beta1 prevented resting cells to enter in the cell cycle when stimulated with growing medium (newborn calf serum plus a mixture of five hormones) but not when TSH (thyroid stimulating hormone) plus IGF-1 (Insulin-like growth factor I) were used as mitogens. Both stimuli increased the levels of cyclins D1, D3 and E but TGF-beta1 had a greater effect in decreasing these cyclin levels in growing-medium stimulated cells than in TSH + IGF-1. This suggests that for FRTL-5 cells, the content of these cyclins must exceed a threshold to progress through the cell cycle. TGF-beta1 induced apoptosis in quiescent cells, accompanied by a reduction in p27 protein levels and an increase in c-myc expression. Interestingly, TGF-beta1-induced variations in prothymosin alpha and c-myc mRNA levels were not correlated. TGF-beta1 always promoted an increase of p15 mRNA levels. In summary, our results point to the fact that TGF-beta1 could play a physiological role in the control of thyroid growth through the modification of cell cycle regulatory proteins.

Expression of epithelial phenotype is enhanced by v-Ha-ras in rat endometrial cells immortalized by SV40 T antigen.

To study the interplay of steroid hormones and oncogenes in the control of endometrial cell proliferation and differentiation we have generated cell lines derived from rat endometrium by expressing the immortalizing oncogenes adeno E1A or SV40 large T antigen. These lines are positive for mesenchymal markers and contain very few characteristic epithelial proteins. Cell lines expressing a temperature-sensitive mutant of SV40 T antigen exhibit a temperature-dependent morphology and growth behavior, but do not manifest an epithelial phenotype at the non-permissive temperature. Cell lines additionally infected with retroviral vectors carrying the v-Ha-ras oncogene (p21rasArg-12) no longer express collagen type III and recover part of their epithelial potential by expressing cytokeratins and/or cadherin E. Some of these cells also express characteristic decidual marker proteins such as desmin, whereas others express glandular epithelial markers such as uteroglobin. Uteroglobin mRNA levels in these cells are increased by glucocorticoids. The parental temperature-sensitive cells do not contain progesterone receptor but become positive for progesterone receptor at the permissive temperature after infection with the v-Ha-ras-expressing retrovirus. Our results indicate that there is a fluent transition and overlapping between mesenchymal, glandular epithelial and decidual phenotypes of endometrial cells, suggesting that these three cell types are derived from the same stem/precursor cells. The v-Ha-ras oncogene product appears to act on the differentiation pathway at an early step prior to the distinction between decidual and glandular epithelial lineage.

Regulation of Pit-1 expression by ghrelin and GHRP-6 through the GH secretagogue receptor.

GH secretagogues are an expanding class of synthetic peptide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their own specific receptor, the GH-secretagogue receptor. The cloning of the receptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambiguously demonstrated the existence of a physiological system that regulates GH secretion. Somatotroph cell-specific expression of the GH gene is dependent on a pituitary-specific transcription factor (Pit-1). This factor is transcribed in a highly restricted manner in the anterior pituitary gland. The present experiments sought to determine whether the synthetic hexapeptide GHRP-6, a reference GH secretagogue compound, as well as an endogenous ligand, ghrelin, regulate pit-1 expression. By a combination of Northern and Western blot analysis we found that GHRP-6 elicits a time- and dose-dependent activation of pit-1 expression in monolayer cultures of infant rat anterior pituitary cells. This effect was blocked by pretreatment with actinomycin D, but not by cycloheximide, suggesting that this action was due to direct transcriptional activation of pit-1. Using an established cell line (HEK293-GHS-R) that overexpresses the GH secretagogue receptor, we showed a marked stimulatory effect of GHRP-6 on the pit-1 -2,500 bp 5'-region driving luciferase expression. We truncated the responsive region to -231 bp, a sequence that contains two CREs, and found that both CREs are needed for GHRP-6-induced transcriptional activation in both HEK293-GHS-R cells and infant rat anterior pituitary primary cultures. The effect was dependent on PKC, MAPK kinase, and PKA activation. Increasing Pit-1 by coexpression of pCMV-pit-1 potentiated the GHRP-6 effect on the pit-1 promoter. Similarly, we showed that the endogenous GH secretagogue receptor ligand ghrelin exerts a similar effect on the pit-1 promoter. These data provide the first evidence that ghrelin, in addition to its previously reported GH-releasing activities, is also capable of regulating pit-1 transcription through the GH secretagogue receptor in the pituitary, thus giving new insights into the physiological role of the GH secretagogue receptor on somatotroph cell differentiation and function.

GDNF and RET-gene expression in anterior pituitary-cell types.

Glial cell line-derived neurotrophic factor (GDNF) is expressed in many neuronal and non-neuronal tissues during development as well as in adult animals. GDNF signaling is mediated through a two-component system consisting of the so called GDNF receptor-alfa (GFRalpha1) which binds to GDNF. Thereafter this complex binds to and activates the tyrosine kinase receptor RET. In this work, for the first time, we have characterized the expression of both GDNF and RET in the anterior pituitary. First of all, RT-PCR analysis, Western blot and immunohistochemistry of the whole anterior pituitary showed that GDNF, GFRalpha1 and RET are expressed in this gland. Following double-immunofluorescence of consecutive sections we found GDNF immunoreactivity in most cell types, and it was most abundant in corticotrophs (55%), LH (59%) and FSH-producing cells (81%). In contrast, while the majority of somatotrophs (87%) were stained for RET, no positive immunostaining could be detected in other cell types. Taken together, this data indicate that gonadotrophs and corticotrophs are the main source of GDNF synthesized in the anterior pituitary and that the somatotrophs appears to be their target cell. This study provides direct morphological evidences that GDNF may well be acting in a paracrine-like fashion in the regulation of somatotroph cell growth and/or cell function.

Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland.

Ghrelin, a 28-amino-acid acylated peptide, strongly stimulates GH release and food intake. In the present study, we found that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs but not in corticotrophs or gonadotrophs of rat pituitary. Persistent expression of the ghrelin gene is found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-d-old rats onward, but at 60 d old, the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Taking into account that somatotroph cell function is markedly influenced by thyroid hormones, glucocorticoids, GH, and metabolic status, we also assessed such influence. We found that ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. In conclusion, we have defined the specific cell types that express ghrelin in the rat anterior pituitary gland. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine-like fashion in the regulation of anterior pituitary cell function. In addition, we clearly demonstrate that pituitary ghrelin mRNA levels are age and gender dependent. Finally, we show that pituitary ghrelin mRNA levels are influenced by alteration on thyroid hormone, glucocorticoids, and GH levels but not by fasting, which indicates that the regulation of ghrelin gene expression is tissue specific.

Acipimox-mediated plasma free fatty acid depression per se stimulates growth hormone (GH) secretion in normal subjects and potentiates the response to other GH-releasing stimuli.

Increases in plasma free fatty acids (FFA) inhibit the GH response to a variety of stimuli; however, the role of FFA depression in GH control is far from understood. In the present work, FFA reduction was obtained by the administration to normal subjects of acipimox, a lipid-lowering drug devoid of side-effects. Each subject tested underwent two paired tests. In one, acipimox was administered orally at a dose of 250 mg at -270 min and at a dose of 250 mg at -60 min; in the matched test, placebo was given at similar intervals. To induce GH release, four stimuli acting through different mechanisms were used: pyridostigmine (120 mg, orally) at -60 min, GHRH (1 microgram/kg, iv) at 0 min, GH-releasing peptide (GHRP-6; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; 1 microgram/kg, iv) at 0 min, and finally, GHRH plus GHRP-6 at the same doses at 0 min. GH secretion was analyzed as the area under the secretory curve (AUC; mean +/- SE, micrograms per L/120 min). Acipimox pretreatment alone (n = 6) induced a reduction in FFA levels compared with placebo treatment. The FFA reduction led to a sustained GH secretion that increased from 2.4 +/- 1.8 micrograms/L at -120 min to 14.2 +/- 4.0 at 120 min. The GH AUC for placebo was 266 +/- 100, and that for acipimox was 1781 +/- 408 (P < 0.05). In the pyridostigmine-treated group (n = 6), the acipimox-pyridostigmine AUC (2046 +/- 323) was higher (P < 0.05) than the placebo-pyridostigmine AUC (764 +/- 101), but was not different from the AUC of acipimox alone. Previous FFA reduction nearly doubled the GHRH-mediated GH secretion (n = 6; placebo-GHRH AUC, 1817 +/- 365; acipimox-GHRH test, 3228 +/- 876; P < 0.05). A similar enhancement was observed when the stimulus employed was GHRP-6 (n = 6; placebo-GHRP-6 AUC, 2034 +/- 295; acipimox-GHRP-6, 4827 +/- 703; P < 0.05). Furthermore, even the most potent GH stimulus known to date, i.e. GHRH plus GHRP-6, was enhanced by the FFA suppression (placebo-GHRH-GHRP-6 AUC, 2034 +/- 277; acipimox-GHRH-GHRP-6, 5809 +/- 758; P < 0.05). The enhancing effect of lowering FFA levels was additive regardless of the stimulus employed. These results indicate that 1) FFA reduction per se stimulates GH secretion with a delayed time of action; 2) FFA reduction enhanced in an additive manner the GH secretion elicited by such different stimuli as pyridostigmine, GHRH, and GHRP-6; and 3) the observation that FFA reduction enhanced the response to the most potent GH stimulus, GHRH plus GHRP-6, suggests that FFA suppression acts by a separate mechanism. FFA reduction may have value in the clinical setting for assessing GH reserve.

Impaired growth hormone secretion in obese subjects is partially reversed by acipimox-mediated plasma free fatty acid depression.

GH secretion in response to provocative stimuli is blunted in obese patients. On the other hand, increases in plasma free fatty acids (FFA) inhibit the GH response to a variety of stimuli, and FFA levels in plasma are increased with obesity. To ascertain whether FFA might be responsible for the GH secretory alterations of obesity, we studied spontaneous and stimulated GH secretion in 31 obese patients after FFA reduction by acipimox, a lipid-lowering drug devoid of serious side-effects. Each subject underwent two paired tests. In one, acipimox was administered orally at a dose of 250 mg at -270 min and at a dose of 250 mg at -60 min; in the matched test, placebo was given at similar intervals. To induce GH release, three stimuli acting through different mechanisms were used: pyridostigmine (60 mg, orally, at -60 min), GHRH (100 micrograms, iv, at 0 min), and GHRH plus GH-releasing peptide (GHRP-6; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2; both at a dose of 100 micrograms, iv, at 0 min). GH secretion was analyzed as the area under the secretory curve (AUC; mean +/- SE; micrograms per L/60 min). Acipimox pretreatment alone (n = 13) induced a large reduction in FFA levels compared with placebo treatment. The FFA reduction led to a slight GH rise (AUC, 123 +/- 47), not different from that in the placebo group (61 +/- 15). In the pyridostigmine-treated group (n = 6), the acipimox-pyridostigmine AUC (408 +/- 107) was significantly higher (P < 0.05) than that in the placebo-pyridostigmine group (191 +/- 25). Furthermore, the GHRH-mediated (n = 6) AUC of GH secretion in the placebo test (221 +/- 55) was tripled by FFA reduction due to acipimox, with an AUC of (691 +/- 134; P < 0.05). Even the most potent GH stimulus known to date, i.e. GHRH plus GHRP-6, was enhanced by FFA suppression. In fact, the placebo-GHRH-GHRP-6 AUC was 1591 +/- 349, lower (P < 0.05) than that in the acipimox-GHRH-GHRP-6 test (2373 +/- 242). The enhancing effects of FFA lowering on GHRH-mediated and GHRH- plus GHRP-6-mediated GH release were synergistic. These results indicate that in obese subjects, unlike normal weight subjects. FFA reduction per se does not stimulate GH secretion. A reduction in FFA with acipimox, however, increased pyridostigmine-. GHRH-, and even GHRH- plus GHRP-6-mediated GH release, suggesting that FFA reduction operates through a different mechanism from that of these three stimuli. The abnormally high FFA levels may be a contributing factor for the disrupted GH secretory mechanisms in obesity.

Regulation of thymosin beta 4 mRNA levels during cell proliferation.

The levels of thymosin beta 4 mRNA were studied throughout the cell cycle of NIH 3T3 cells. In serum deprived, quiescent cells, the levels of thymosin beta 4 were undetectable; after serum restoration, the cells were induced to proliferate and we found a pronounced increase in thymosin beta 4 mRNA levels at the G1/S transition. Thymosin beta 4 mRNA was induced even in the presence of cycloheximide. On the other hand, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake-off after nocodazole arrest or a double thymidine block did not show any variation in the levels of thymosin beta 4 mRNA when they progressed synchronously through the cycle. In conclusion, the present data indicate that the thymosin beta 4 gene is regulated by cell proliferation but it is not a cell cycle-regulated gene. Finally, we studied thymosin beta 4 mRNA stability by inhibiting thymosin beta 4 gene transcription with actinomycin D. Our results suggest that thymosin beta 4 mRNA has a pronounced stability, a fact that might be relevant to account for the presence of thymosin beta 4 in enucleated cells like platelets.

Prothymosin alpha mRNA is expressed in competent and proliferating rat thyroid cells (FRTL-5) but is not sufficient to elicit cell progression through the cell cycle.

Using flow cytometry we observed the effects that different hormonal treatments had on the progression of rat thyroid (FRTL-5) cells through the cell cycle. The absence of hormones or the addition of TSH (6 mU/ml) did not induce DNA synthesis; however, the addition of IGF-I (30 ng/ml) promoted cell proliferation. The number of cells recruited by IGF-I was lower than when IGF-I and TSH were used. We therefore concluded that we had a model with three different types of cells: (1) quiescent cells, cells cultured in the absence of hormones, considered to be G0-arrested cells, (2) competent cells, TSH-treated cells that did not proliferate (being arrested in a cycle phase different from G0) and (3) actively proliferating cells, cells treated with TSH plus IGF-I. Prothymosin alpha (PTA) mRNA levels were almost undetectable in cells cultured without hormones at all times studied, i.e. 8, 14 and 24 h. On the contrary, TSH and/or IGF-I greatly increased PTA mRNA. These data indicate that G0-arrested quiescent cells do not express PTA mRNA and that PTA mRNA is induced when FRTL-5 cells are committed to proliferate by the addition of TSH, in spite of being arrested by the lack of IGF-I. We therefore conclude that PTA mRNA expression may be an event that is necessary for cells to proliferate, but that it is not sufficient for the promotion of cell progression through the cell cycle.

Effects of TGF-beta1 on prolactin synthesis and secretion: an in-vitro study.

The hypothalamus exerts a predominantly inhibitory influence on prolactin secretion through dopamine. In addition, the expression of anterior pituitary hormone-gene products are regulated by intrapituitary growth factors. In particular, TGF-beta1 produced in the pituitary regulates lactotroph cell proliferation and prolactin gene-expression. This study characterized the regulation of in-vitro prolactin synthesis and secretion by TGF-beta1 using rat anterior pituitary cells in monolayer culture. Furthermore, we studied the interaction of TGF-beta1 with other signals involved in the neuroregulation of prolactin secretion, such as dopamine and TRH, as well as the importance of different signal transduction pathways in this response. TGF-beta1 inhibited prolactin secretion in a time- and concentration-dependent manner, with half-maximal inhibition occurring at the range of 15-30 pM. The inhibitory effect was observed after 4 h, being maximal after 4 days of exposure of the cells to the peptide. This inhibitory effect was mimicked by TGF-beta2 but not by inhibin, and was not influenced by oestrogens, being similar in male, normal female or oestradiol-treated rats. Prolonged pretreatment of the cells with TGF-beta1(4 days) did not modify GH or TSH secretion nor dopamine-induced inhibition of prolactin secretion, and blunted prolactin responses to TRH, Forskolin, But2-cAMP and to the calcium ionophore A23187. The effect observed after long-term treatment (24 h to 4 days) is essentially caused by a decrease in prolactin synthesis, since TGF-beta1 inhibited prolactin mRNA levels and de novo prolactin protein synthesis. However, in the short term (up to 12 h) TGF-beta1 inhibition of prolactin secretion was associated with an increase in intracellular prolactin content, dissecting a dual mechanism of action of TGF-beta1. The short-term TGF-beta1 effect did not modify Erk-2 phosphorylation, basal or TRH-induced increase in intracellular calcium concentration, but blunted basal and forskolin stimulated cAMP levels. But2-cAMP replacement did not revert the inhibition of prolactin secretion. However, pertussis toxin was able to recover a large percentage of TGF-beta1-induced inhibition of prolactin secretion. This study indicates that TGF-beta1 plays a crucial role as a modulator of lactotroph function, inhibiting prolactin biosynthesis after long-term treatment, as well as, after short-term exposure prolactin secretion at the level of the secretory process, through a mechanism pertussis toxin sensitive but independent of Erk-2 phosphorylation, calcium concentrations or intracellular cAMP.

Effect of neurotensin on growth hormone release in vivo.

In order to investigate the mechanisms involved in the in vivo Growth Hormone (GH) response to Neurotensin (NT) we assessed the influence of estrogen status as well as the effect of passive immunization with antisomatostatin and anti-Growth Hormone-Releasing Hormone (GHRH) on NT-induced GH secretion in pentobarbital anesthetized rats. We found that, contrary to GH responses to GHRH, estrogen-treated rats (one single injection of 200 micrograms s.c. of estradiol valerate, 3 days before the experiment), exhibited markedly increased GH responses to different doses of NT (7.5, 15 and 30 micrograms/kg, i.v.). The stimulatory effect of NT (30 micrograms/kg) on estrogen-treated rats was similar in rats that received normal rabbit serum or passively immunized with antisomatostatin or anti-GHRH serum. In conclusion, estrogens play a facilitatory role on NT-induced GH release in the rat, which is exerted through a mechanism independent of hypothalamic GHRH or somatostatin release.

Direct promoter induction of p19Arf by Pit-1 explains the dependence receptor RET/Pit-1/p53-induced apoptosis in the pituitary somatotroph cells.

Somatotrophs produce growth hormone (GH) and are the most abundant secretory cells of the pituitary. Somatotrophs express the transcription factor Pit-1 and the dependence receptor RET, its co-receptor GFRa1 and ligand GDNF. Pit-1 is a transcription factor essential for somatotroph proliferation and differentiation and for GH expression. GDNF represses excess Pit-1 expression preventing excess GH. In the absence of GDNF, RET behaves as a dependence receptor, becomes intracellularly processed and induces strong Pit-1 expression leading to p53 accumulation and apoptosis. How accumulation of Pit-1 leads to p53 expression is unknown. We have unveiled the relationship of Pit-1 with the p19Arf gene. There is a parallel correlation of RET processing, Pit-1 increase and ARF protein and mRNA expression. Interfering the pathway with RET, Pit-1 or p19Arf siRNA blocked apoptosis. We have found a Pit-1 DNA-binding element within the ARF promoter. Pit-1 directly regulates the CDKN2A locus and binds to the p19Arft promoter inducing p19Arf gene expression. The Pit-1-binding element is conserved in rodents and humans. RET/Pit-1 induces p19Arf/p53 and apoptosis not only in a somatotroph cell line but also in primary cultures of pituitary somatotrophs, where ARF siRNA interference also blocks p53 and apoptosis. Analyses of the somatotrophs in whole pituitaries supported the above findings. Thus Pit-1, a differentiation factor, activates the oncogene-induced apoptosis (OIA) pathway as oncogenes exerting a tight control in somatotrophs to prevent the disease due to excess of GH (insulin-resistance, metabolic disease, acromegaly).

Identification of a paired box gene 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARgamma) rearrangement mosaicism in a patient with an autonomous functioning follicular thyroid carcinoma bearing an activating mutation in the TSH receptor.

Our main objective was to search for mutations in candidate genes and for paired box gene 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARgamma) rearrangement in a well-differentiated angioinvasive follicular thyroid carcinoma (FTC) causing hyperthyroidism. DNA and RNA were extracted from the patient's thyroid tumor, as well as 'normal' thyroid tissue, and from peripheral blood lymphocytes (PBLs) of the patient, her daughter, and two siblings. Nuclear isolation was extracted from the patient's tumor, 'normal' thyroid tissue, PBLs, and uterine leiomyoma tissue. TSH receptor (TSHR), RAS, and BRAF genes were sequenced. We searched for PAX8-PPARgamma in thyroid, PBL, and uterine leiomyoma samples from the patient and family members. Proliferative effects of detected mutants on non-transformed human thyrocytes cultures. An activating TSHR mutation, M453T, was detected in the tumor. PAX8 (exons 1-8+10)-PPARgamma was found in all tested patient's tissues. A second rearrangement, PAX8 (exons 1-8)-PPARgamma, was detected in the patient's normal thyroid tissue. Under deprived medium condition, co-transfection of PAX8-PPARgamma and TSHR-M453T dramatically increased the number of thyrocytes, an effect that it was not observed with TSHR wild-type (WT); under complete medium conditions, co-transfection of PAX8-PPARgamma with either TSHR-M453T or TSHR-WT inhibited cell proliferation. We report a patient with hyperthyroidism due to a FTC bearing an activating TSHR mutation and PAX8-PPARgamma rearrangements. PAX8-PPARgamma was present as a mosaicism affecting tissues from endodermal and mesodermal origin. PAX8-PPARgamma and TSHR-M453T inhibited or promoted thyrocyte proliferation depending on medium conditions. The activating TSHR mutation could promote in vivo FTC development in PAX8-PPARgamma-positive thyrocytes under poor blood supply with deprivation of growth factors but restraint the tumor growth when growth factors are supplied.

New insights into thyroglobulin pathophysiology revealed by the study of a family with congenital goiter.

CONTEXT: Thyroglobulin (TG) gene mutations cause congenital hypothyroidism (CH) with goiter. A founder effect has been proposed for some frequent mutations. Mutated proteins have a defect in intracellular transport causing intracellular retention with ultrastructural changes that resemble an endoplasmic reticulum storage disease. OBJECTIVE: To reveal new aspects of thyroglobulin pathophysiology through clinical, cellular, molecular, and genetic studies in a family presenting with CH due to TG mutations from Galicia, an iodine-deficient area of Spain. DESIGN: The included clinical evaluation of family members, DNA sequencing for TG gene mutation and haplotyping analysis, ultrastructural analysis of thyroid tissue specimens from affected subjects, analysis of effects of mutations found on TG gene transcription, and in vitro studies of cellular production and secretion of mutated proteins. SETTING: Locations included primary care and university hospitals. RESULTS: Family members with CH, mental retardation, and goiter were compound heterozygous for c.886C-->T (p.R277X) and g.IVS35+1delG. For c.886C-->T, a founder effect cannot be excluded, and its transcription was hardly detectable. g.IVS35+1delG caused an in-frame deletion in exon 35 and produced a protein that, although synthesized, could not be secreted. Ultrastructural analyses showed morphological changes consistent with an endoplasmic reticulum storage disease. CONCLUSION: The shorter thyroglobulin resulting from the novel g.IVS35+1delG was retained within the endoplasmic reticulum of thyrocytes, and together with p.R227X caused severe hypothyroidism with goiter. p.R277X, the most commonly described TG mutation, is caused by a TG exon-7 highly mutation-prone region, and the possibility that some cases were introduced to South America from Galicia cannot be excluded.