Neurocognitive and familial moderators of psychiatric risk in velocardiofacial (22q11.2 deletion) syndrome: a longitudinal study.

BACKGROUND: Although risk for psychosis in velocardiofacial (22q11.2 deletion) syndrome (VCFS) is well established, the cognitive and familial factors that moderate that risk are poorly understood. METHOD: A total of 75 youth with VCFS were assessed at three time points, at 3-year intervals. Time 1 (T1) psychiatric risk was assessed with the Behavior Assessment System for Children (BASC). Data reduction of BASC scores yielded avoidance-anxiety and dysregulation factors. Time 2 (T2) neuropsychological and family function and time 3 (T3) prodromal/overt psychosis were assessed. Poisson regression models tested associations between T3 positive prodromal symptoms/overt psychosis and T1 psychiatric risk, T2 cognitive and familial factors, and their interactions. RESULTS: T1 avoidance-anxiety ratings predicted T3 prodromal/overt psychosis. T2 verbal learning scores moderated this association, such that individuals with low avoidance-anxiety scores and stronger verbal learning skills were the least likely to demonstrate prodromal/overt psychosis at T3. Low scores on a T2 visual vigilance task also predicted T3 prodromal/overt psychosis, independently of the effect of T1 avoidance-anxiety scores. T1 dysregulation scores did not predict T3 prodromal/overt psychosis in a linear manner. Instead, the association between dysregulation and prodromal/overt psychosis was amplified by T2 levels of family organization, such that individuals with low dysregulation scores and low family organization scores were the most likely to exhibit T3 prodromal/overt psychosis. CONCLUSIONS: Significant moderators of psychiatric risk in VCFS include verbal learning skills as well as levels of family organization, carrying implications for early identification and preventative treatment of youth with VCFS at highest risk for psychosis.

Role of glucocorticoid hormones in arginine vasopressin gene regulation.

The mechanism underlying increased AVP synthesis and release in glucocorticoid deficiency is not known. Therefore, the present study was undertaken to investigate whether the mechanism was at the level of AVP gene transcription. The AVP gene promoter contains a consensus GRE, a CRE, and four AP2 sites. To assess the functional importance of these sites, 5' deletions of the AVP promoter were created and transient transfections were performed. Promoter activity in hypothalamic cells transfected with deletions lacking the GRE or both the GRE and CRE exhibited higher activity when compared to longer constructs containing both sites. In neuroblastoma cells, only the deletion lacking the GRE exhibited increased AVP promoter activity over the longer construct. These results are consistent with the idea that glucocorticoids suppress AVP gene expression by acting on a GRE in the AVP promoter region. Further, dexamethasone inhibited AVP promoter activity by >50% in hypothalamic cells transfected with the GRE-containing construct. In conclusion, the data presented here support a central mechanism to explain, at least in part, the nonosmotic increase in AVP with glucocorticoid deficiency.

Signaling pathways responsible for fetal gene induction in the failing human heart: evidence for altered thyroid hormone receptor gene expression.

BACKGROUND: We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). METHODS AND RESULTS: To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of alpha-MHC with increased beta-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-alpha(1), -alpha(2), and -beta(1) isoforms in human left ventricles. In failing left ventricles, TR-alpha(1) was downregulated, whereas TR-alpha(2), a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-beta(1) did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-alpha(1) and -alpha(2) were positively and negatively correlated with those of alpha-MHC, respectively. CONCLUSIONS: We conclude that decreases in TR-alpha(1) and increases in TR-alpha(2) may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.

The effect of thyroid hormone and a long-acting somatostatin analogue on TtT-97 murine thyrotropic tumors.

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Cloning of the mouse somatostatin receptor subtype 5 gene: promoter structure and function.

Somatostatin is a peptide hormone whose actions are mediated by five somatostatin receptor subtypes (sstl-5). In the pituitary, somatostatin inhibits TSH release from thyrotropes and GH release from somatotropes. We have shown that sst5 transcripts and protein are induced by thyroid hormone in TtT-97 thyrotropic tumors. To map sequences responsible for promoter activity in pituitary cells, we cloned the mouse sst5 coding region of 362 amino acids and 12 kb of upstream DNA. Initial transfection studies in TtT-97 or GH3 cells mapped high levels of basal promoter activity to a 5.6-kb fragment upstream of the translational start, whereas shorter genomic fragments had low activity. To identify the transcriptional start site we used 5' RACE with TtT-97 poly A+ RNA and a sst5 antisense coding region primer. Sequence comparison between the complementary DNA and the gene revealed that the mouse sst5 gene contains 3 exons and 2 introns. The entire coding region was contained in exon 3. Two differently sized RACE products demonstrated alternate exon splicing of two untranslated exons in TtT-97 cells. A promoter fragment from -290/+48 linked to a luciferase reporter demonstrated 600- and 900-fold higher activity over a promoterless control in GH3 mammosomatotropes and TtT-97 thyrotropes, respectively, whereas a larger fragment extending to -6400 exhibited no additional promoter activity. Cloning of the sst5 gene will facilitate the mapping of basal and regulated responses at the transcriptional level.

An upstream regulator of the glycoprotein hormone alpha-subunit gene mediates pituitary cell type activation and repression by different mechanisms.

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.

Reverse transcription polymerase chain reaction analysis of pituitary hormone, Pit-1 and steroidogenic factor-1 messenger RNA expression in pituitary tumors.

Pit-1 is a transcription factor that appears early in embryonic pituitary gland formation and is necessary for the development of somatotropes, lactotropes and thyrotropes. Steroidogenic factor-1 (SF-1) is another early appearing transcription factor that is involved in the development of gonadotropes. In this study we have compared RT-PCR analysis of hormone mRNA with traditional IHC for classification of 27 pituitary tumors and have evaluated the correlation of Pit-1 and SF-1 mRNA with hormone mRNA. RT-PCR detected concordant hormone mRNA in 100% of GH IHC positive, 100% of PRL IHC positive, 33% of TSH IHC positive, and 93% of gonadotropin IHC positive tumors. IHC, however, was concordant in only 71% of GH mRNA positive, 78% of PRL mRNA positive, 17% of TSH beta mRNA positive, and 76% of FSH beta mRNA positive tumors. Pit-1 mRNA was positive in 87% of tumors in which mRNA for GH, PRL or TSH beta was detected and in only 17% of GH, PRL and TSH beta mRNA negative tumors. SF-1 mRNA was positive in 94% of tumors in which mRNA for FSH beta was present and in no FSH beta mRNA negative tumors. We conclude that RT-PCR analysis of hormone mRNA may be more sensitive than traditional hormone IHC for classification of pituitary tumors. Furthermore, tumor Pit-1 mRNA positively correlates with GH, PRL and TSH beta mRNA while tumor SF-1 mRNA correlates well with FSH beta mRNA. Combined analysis of hormone and transcription factor mRNA in pituitary tumor tissue may therefore be a more meaningful approach to pituitary tumor characterization.

Functional interactions of an upstream enhancer of the mouse glycoprotein hormone alpha-subunit gene with proximal promoter sequences.

Transcription of the glycoprotein hormone alpha-subunit gene in the pituitary is governed by different promoter elements in thyrotropes and gonadotropes. We recently identified an upstream enhancer that directs a high level of cell type specific expression in transgenic mice and stimulates proximal promoter activity in cultured alphaTSH and alphaT3 cells. To assess the contribution of promoter sequences that functionally interact with the enhancer, we mutated two proximal elements shown to be important in both thyrotrope and gonadotrope cells. Disruption of the pituitary glycoprotein hormone basal element (PGBE), which binds a LIM homeodomain protein, resulted in a decrease in basal promoter activity in both alphaTSH and alphaT3 cells. Enhancer function was completely abolished by the PGBE site mutation in alphaT3 gonadotropes, whereas some stimulatory activity remained in alphaTSH thyrotropes. Mutation of the gonadotrope specific element (GSE), which binds SF1 and is important for basal activity in gonadotropes and TRH response in thyrotropes, resulted in declines in basal and enhanced promoter activity only in alphaT3 cells and not in alphaTSH cells. Despite this decrease in enhanced activity, the GSE mutated promoter still retained some enhancer stimulated activity, suggesting that the PGBE site still functionally interacts in the absence of an intact GSE. This mutation had no effect in alphaTSH cells. These data suggest that although the enhancer works in both cell types it exhibits cell type specific functional characteristics.

Cell-specific expression of the mouse glycoprotein hormone alpha-subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells.

The glycoprotein hormone alpha-subunit gene is expressed and differentially regulated in pituitary gonadotropes and thyrotropes. Previous gene expression studies suggested that cell specificity may be regulated by distinct DNA elements. We have identified an enhancer region between -4.6 and -3.7 kb that is critical for high level expression in both gonadotrope and thyrotrope cells of transgenic mice. Fusion of the enhancer to -341/+43 mouse alpha-subunit promoter results in appropriate pituitary cell specificity and transgene expression levels that are similar to levels observed with the intact -4.6 kb/+43 construct. Deletion of sequences between -341 and -297 resuited in a loss of high level expression and cell specificity, exhibited by ectopic transgene activation in GH-, ACTH-, and PRL-producing pituitary cells as well as in other peripheral tissues. Consistent with these results, transient cell transfection studies demonstrated that the enhancer stimulated activity of a -341/+43 alpha-promoter in both alphaTSH and alphaT3 cells, but it did not enhance alpha-promoter activity significantly in CV-1 cells. Removal of sequences between -341 and -297 allowed the enhancer to function in heterologous cells. Loss of high level expression and cell specificity may be due to loss of sequences required for binding of the LIM homeoproteins or the alpha-basal element 1. These data demonstrate that the enhancer requires participation by both proximal and distal sequences for high level expression and suggests that sequences from -341 to -297 are critical for restricting expression to the anterior pituitary.

Activation of the glycoprotein hormone alpha-subunit gene promoter in thyrotropes.

Pituitary expression of the glycoprotein hormone alpha-subunit gene localizes to thyrotropes and gonadotropes. Factors that activate the alpha-subunit promoter exclusively in thyrotropes have not been described. In the current studies, alpha-subunit promoter activity was compared in alphaTSH thyrotropic and alphaT3 gonadotropic cells. Deletional analysis revealed a more important contribution of the -453/-381 region in thyrotropic cells and the -341/-297 region in gonadotropic cells, while other deletions had similar effects. Mutational analysis revealed some regions that were functionally similar, while others were active only in thyrotropic cells, such as the regions - 434/ - 421, - 398/ - 385, - 376/- 364 and - 363/- 351, that decreased activity by approximately 2-fold each. Combined mutation of the regions - 434/ - 421, - 398/ - 385 and - 376/ - 364 decreased activity by 5-fold in thyrotropic cells and not at all in gonadotropic cells. Cotransfection with Ptx1 in CV-1 cells resulted in a strong stimulation of alpha-subunit promoter activity, that was significantly diminished with mutation of the - 398/ - 385 region that disrupts the Ptx1 binding site, suggesting that this factor may play a role in thyrotrope-specific activation. This analysis provides basic information important to identify common and unique factors contributing to the cell-specific expression of the alpha-subunit gene.

Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.

Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10[-2] U beta-galactosidase [beta-gal]), but not the mutated BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP secretion (3.0 +/- 1.4 pg/10[-2] U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13.5 +/- 4.0 pg/10[-2] U beta-gal). When a stop codon was inserted into the wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10[-2] U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05). In summary, (1) both VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region diminishes but does not abolish AVP processing and secretion; and (3) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild-type gene precursor. Thus, the CDI in BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.

Msx1 is present in thyrotropic cells and binds to a consensus site on the glycoprotein hormone alpha-subunit promoter.

Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alphaTSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alphaTSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alphaTSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the alpha-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an alpha-subunit promoter construct decreased by approximately 50% in transfected thyrotrope (alphaTSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.

Progesterone regulated expression of flavin-containing monooxygenase 5 by the B-isoform of progesterone receptors: implications for tamoxifen carcinogenicity.

Progesterone is a key developmental, proliferative, and differentiative hormone in the breast and endometrium, and it can accelerate carcinogenesis in the mammary gland epithelium. In the breast and uterus, progesterone acts through two coexpressed isoforms of progesterone receptors, the B- and A-receptors. To study the function of each isoform in isolation, we previously constructed two breast cancer cell lines that stably and independently express either B-receptors (YB cells) or A-receptors (YA cells). In the present study, YA or YB cells were left untreated, or were treated with the synthetic progestin R5020, and the messages present in each cell line under the two conditions were analyzed by differential display. Two message species are described that are regulated only by B-receptors. One of these is regulated in a ligand-independent manner. A third set of messages, encoding flavin-containing monooxygenase 5 (FMO5), was induced by R5020 only in YB cells. A-receptors appear to be inhibitory. FMOs are involved in the metabolic activation of drugs and xenobiotic compounds, including the antiestrogen tamoxifen, to carcinogenic intermediates. It is possible, therefore, that by upregulating the levels of FMO5, progesterone enhances the carcinogenicity of tamoxifen in target tissues that overexpress progesterone B-receptors.

Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter.

The molecular determinants governing cell-specific expression of the thyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not well understood. The P1 region of the mouse TSHbeta promoter (-133 to -88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors. In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells. The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody. When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSHbeta promoter activity 8.5-fold, while each factor alone had a minimal effect. Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution. In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.

Reconstitution of triiodothyronine inhibition in non-triiodothyronine-responsive thyrotropic tumor cells using transfected thyroid hormone receptor isoforms.

The triiodothyronine (T3) inhibitory effect on the thyrotropin (TSH)beta- and alpha-subunit genes is believed to be mediated by binding of T3 to specific nuclear receptors that are present in various isoforms. alphaTSH cells, which are derived from a pure alpha-subunit secreting thyrotropic tumor, contain the same nuclear factors that are important for alpha-subunit gene expression in TSH-expressing T3-responsive thyrotropic cells (TtT97). However, as in the parent tumor, alpha-subunit expression in alphaTSH cells was not inhibited by T3, despite the presence of high-affinity nuclear T3 receptors (TRs) with a similar number of sites per cell as in TtT97. When transcripts coding for the different TR isoforms from the MGH101A tumor were analyzed by Northern blot, TR alpha1 was present, as well as the non-T3-binding variant alpha2, but transcripts encoding the opposite strand Rev-ErbAa were not detectable and neither TR beta1 nor TR beta2 mRNAs were detectable, whereas all these transcripts were detectable in TtT97 tumors. Similar findings were observed in alphaTSH cells, where TR beta1 transcripts were barely detectable in Northern blots and TR beta2 transcripts were detectable only by RT-PCR. The TR beta gene locus is present and unrearranged in the tumor genome. In transient transfection studies conducted in alphaTSH cells overexpression of either TR beta1, TR beta2, or TR alpha1 reconstituted T3-inhibition of the alpha-subunit promoter down to 40% to 50% of control. We conclude that the relative lack of TR beta gene expression correlates with unresponsiveness to T3. The alphaTSH cell line represents a unique model in which to dissect the mechanism of T3 inhibition.

The thyrotrope-restricted isoform of the retinoid-X receptor-gamma1 mediates 9-cis-retinoic acid suppression of thyrotropin-beta promoter activity.

TSHbeta is a subunit of TSH that is uniquely expressed and regulated in the thyrotrope cells of the anterior pituitary gland. Thyroid hormone receptors (TR) are known to mediate T3 suppression of TSHbeta gene expression at the level of promoter activity. The role of other nuclear receptors in regulation of this gene is less clearly defined. Retinoid X receptors (RXR) are a family of nuclear transcription factors that function both as 9-cis-retinoic acid (RA) ligand-dependent receptors and heterodimeric partners with TR and other nuclear receptors. Recently, the RXR isoform, RXRgamma, has been identified in the anterior pituitary gland and found to be restricted to thyrotrope cells within the pitutiary. In this report, we have further characterized the distribution of RXRgamma1, the thyrotrope-restricted isoform of RXRgamma, in murine tissues and different cell types. We have found that RXRgamma1 mRNA and protein are expressed in the TtT-97 thyrotropic tumor, but not the thyrotrope-variant alphaTSH cells or somatotrope-derived GH3 cells. Furthermore, we have studied the effects of RXRgamma1 on TSHbeta promoter activity and hormone regulation in these pituitary-derived cell types. Both T3 and 9-cis-RA independently suppressed promoter activity in the TtT-97 thyrotropes. Interestingly, the combination of ligands suppressed promoter activity more than either alone, indicating that these hormones may act cooperatively to regulate TSHbeta gene expression in thyrotropes. The RXRgamma1 isoform was necessary for the 9-cis-RA-mediated suppression of TSHbeta promoter activity in alphaTSH and GH3 cells, both of which lack this isoform. RXRbeta, a more widely distributed isoform, did not mediate these effects. Finally, we showed that the murine TSHbeta promoter region between -200 and -149 mediated a majority of the 9-cis-RA suppression of promoter activity in thyrotropes. This region is distinct from the T3-mediated response region near the transcription start site. These data suggest that retinoids can mediate TSHbeta gene regulation in thyrotropes and the thyrotrope-restricted isoform, RXRgamma1, is required for this effect.

Thyroid hormone-induced expression of specific somatostatin receptor subtypes correlates with involution of the TtT-97 murine thyrotrope tumor.

Following the protracted hypothyroid state, treatment with thyroid hormone will induce a decline in TSH and reduce thyrotrope hyperplasia. Somatostatin is a hypothalamic peptide that has been implicated in the negative regulation of TSH secretion in the thyrotrope. Moreover, analogs of native somatostatin have potent TSH-reducing and growth-retarding effects on human thyrotropinomas. The TtT-97 tumor is an in vivo murine thyrotropic model that has retained its physiological response to thyroid hormone. This study investigates the regulation of somatostatin receptor subtypes in this tumor. TtT-97 tumors, actively growing in hypothyroid mice, did not express any significant somatostatin receptor messenger RNA (mRNA) or protein. T4 administration resulted in a reduction in TSH beta mRNA expression and a marked degree of tumor involution. Analysis of residual tumors from thyroid hormone-treated mice showed the specific up-regulation of SSTR1 and SSTR5 mRNA subtypes and the appearance of abundant, high affinity SSTR receptor-binding sites within the tumor. Thus, the TtT-97 tumor provides a thyrotrope-specific model in which to study the regulation of somatostatin receptor subtypes by thyroid hormone and correlate this expression with both antisecretory and antiproliferative effects.

Insulin internalization in the absence of the insulin receptor tyrosine kinase domain is insufficient for mediating intracellular biological effects.

The intracellular portion of the human insulin receptor (hIR) beta-subunit contains distinct functional domains including the exon 16-encoded juxtamembrane (JM) domain that mediates endocytosis, and the tyrosine kinase (TK) domain that mediates insulin's metabolic and mitogenic actions. To explore the functional relationship between these domains and to determine the role of insulin internalization in insulin action, we constructed and studied the endocytic and signaling properties of an hIR mutant truncated at Glu-1012. This truncation removes the carboxyl-terminal 343 amino acids containing essentially all of the TK domain but leaves behind the exon 16-encoded JM domain that is necessary for endocytosis, plus an additional 35 amino acids downstream. The wild-type (hIR-WT) and mutant (hIR delta 343) receptors were stably expressed in CHO cells and their abilities to mediate various insulin-stimulated functions were comparatively analyzed. In cells expressing hIR-WT, insulin markedly enhanced tyrosine phosphorylation of the beta-subunit and of the endogenous 185 kDa substrate whereas these effects were completely absent in cells expressing hIR delta 343. The hIR delta 343 receptors retained the ability to internalize a significant amount of surface-bound insulin at 37 degrees C. However, they were unable to mediate either the short or long-term biological effects of insulin as determined by assaying insulin-stimulated glucose uptake (assessed by 2-deoxyglucose uptake), protooncogene expression (measured by Northern blot analysis of c-fos mRNA) and DNA synthesis (measured by 3H-thymidine incorporation). These results indicate that the hIR beta-subunit JM and TK domains can be functionally uncoupled, and that insulin internalization in the absence of hIR TK domain and kinase activity is insufficient for mediating intracellular insulin action.

Osmotic and non-osmotic regulation of arginine vasopressin (AVP) release, mRNA, and promoter activity in small cell lung carcinoma (SCLC) cells.

Arginine vasopression (AVP) is synthesized in the magnocellular neurons of the hypothalamus and stored in the posterior pituitary. It has been shown that hypothalamic AVP mRNA is increased during experimental stimulation of osmotic and non-osmotic stimulation of AVP release. The mechanisms underlying the stimulation of AVP biosynthesis in these conditions are not known. The present study was, therefore, performed to measure AVP release, AVP mRNA level, and AVP gene promoter activity during osmotic and non-osmotic stimulation of AVP secretion in the small cell lung carcinoma (SCLC) cells. AVP release was measured by radioimmunoassay, steady state levels of AVP mRNA by solution hybridization, and AVP gene promoter activity exhibited by a 1.5 kb 5'-flanking AVP gene fragment fused to a luciferase reporter after SCLC cells were subjected to osmotic or non-osmotic conditions. High media osmolality (330 mOsm) significantly increased AVP release (control (C) 1.42 +/- 0.27 vs. High Osm 3.67 +/- 0.39 pg/2 x 10(6) cells, N = 9, P < 0.002); AVP mRNA (C 173.6 +/- 16.8 vs. High Osm 280.1 +/- 19.4 pg/2 x 10(6) cells, N = 7, P < 0.001); and AVP gene promoter activity (C 1353 +/- 99 vs. High Osm 2026 +/- 134 L.U./10(-4) U beta-gal, N = 8, P < 0.001). Non-osmotic stimulators. 0.1 microM endothelin 3 (ET3), 1 microM angiotensin II (AII), and 10 microM acetylcholine (Ach) significantly increased AVP release; ET3 (C 1.78 +/- 0.20 vs. ET3 6.85 +/- 1.86 pg/2 x 10(6) cells, N = 8, P < 0.02); AII (C 1.29 +/- 0.38 vs. AII 27.80 +/- 7.09 pg/2 x 10(6) cells, N = 5, P < 0.05) and Ach (C 1.14 +/- 0.33 vs. Ach 2.68 +/- 0.58 pg/2 x x10(6) cells, N = 6, P < 0.05). However, only ET3 significantly increased AVP mRNA (C 166.6 +/- 19.6 vs. ET3 254.4 +/- 25.6 pg/p x 10(6) cells, N = 5, P < 0.05) and AVP promoter activity (C 1515 +/- 163 vs. ET3 2389 +/- 342 L.U./10(-4) U beta-gal, N = 6, P < 0.05). To localize the region of the AVP promoter that mediates the osmotic stimulation and the effect of ET3, 5' deletions of the AVP promoter fragments terminating at -532, -211, and -102, was assessed. Only the promoter activity of the 1.5 kb construct, but not the deletion constructs, was significantly increased by ET3 or high osmolality. These results suggest that modulation of AVP gene transcription is, at least in part, responsible for increased AVP synthesis and release in response to osmotic and non-osmotic stimulation, and that the region of 5' flanking sequence between -1500 and -532 contains the elements responsible for the effects.

Thyroid hormone receptor beta2 promoter activity in pituitary cells is regulated by Pit-1.

There are three known thyroid hormone receptor (TR) isoforms that arise from two distinct alpha and beta gene loci. TRalpha1 and TRbeta1 mRNAs are found in many tissues, whereas mRNA for the N-terminal TRbeta2 variant derived from the beta locus is readily detectable only in the pituitary gland and derived cell sources such as GH3 somatotropes and TtT-97 thyrotropes. We previously isolated the genomic region governing expression of the TRbeta2 isoform in thyrotropes and showed that transcription arose from multiple origins within a 400-base pair (bp) region. We now report that the region extending 500 bp upstream of the putative AUG codon (A is +1) contains six areas of interaction with the pituitary-specific transcription factor Pit-1. In addition there are separate areas that bind other factors present in thyrotrope cells. Promoter deletions revealed that removal of regions containing the Pit-1 sites at -456 to -432, -149 to -127, and -124 to -102 progressively decreased TRbeta2 promoter activity in thyrotropes. A more proximal footprinted area from -65 to -19, which accounted for the remaining promoter activity, contained sites that interacted with recombinant Pit-1; however, extracts of TtT-97 thyrotropes, which express Pit-1, footprinted this proximal region with a pattern of protection that differed from that produced by Pit-1. A comparative deletional analysis demonstrated that a shorter region extending only 204 bp from the AUG was sufficient to support TRbeta2 promoter activity in GH3 somatotropes. The more proximal Pit-1 sites, including the area from -53 to -19, whose pattern differed from Pit-1 in thyrotrope extracts, showed protection patterns with GH3 extracts that were indistinguishable from recombinant Pit-1. Site-directed mutagenesis that abrogated binding of both recombinant Pit-1 and Pit-1-containing nuclear extracts revealed that the two Pit-1 sites between -149 and -102 were important for TRbeta2 promoter activity with the more proximal being most critical. Finally, we showed that TRbeta2 promoter activity in alpha-TSH cells, which do not transcribe the endogenous TRbeta2 locus or produce Pit-1 protein, could be reconstituted to a level approaching that seen in expressing TtT-97 thyrotropes by cotransfecting a Pit-1 expression vector. Activation by Pit-1 was dependent on the same Pit-1 sites shown to be important for basal TRbeta2 promoter activity in thyrotropes as constructs lacking them by deletion or mutation were not stimulated by Pit-1.