Rat Growth Hormone Promoter Research Articles

Abstract 1556: Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors.

Abstract Optical imaging of labeled cells is the most prevailing method for cell tracking in mouse models. However, the immunogenicity of xenobiotic reporter genes, such as the commonly used firefly luciferase (ffLuc) and enhanced green fluorescence protein (eGFP), results in inconsistency in cell labeling and tumor progression, preventing their use in host mice with normal immunity. To resolve this issue, we have generated a reporter gene-tolerized transgenic mouse in which an ffLuc-eGFP fusion gene was targeted to the anterior pituitary gland using a rat Growth Hormone promoter (dubbed the “glowing-head” mouse). When ffLuc-eGFP-labeled Lewis Lung Carcinoma (LLC) tissue was subcutaneously transplanted into syngeneic glowing-head mice (GH-c-brd), their non-transgenic litter-mates (WT-c-brd), and immunocompromised Nod-Scid (balb/c) mice, anti-GFP antibody was induced only in the circulation of WT-c-brd. Labeled LLC also exhibited fewer CD4+ T cells but more macrophages in GH-c-brd than in WT-c-brd, suggesting it induced adaptive immunity in the latter. Moreover, GH-c-brd exhibited faster subcutaneous tumor growth and post-resection metastatic progression, and maintained more consistent tumor labeling, as compared to WT-c-brd and even Nod-Scid mice. Interestingly, in an adjuvant chemotherapeutic setting, the immunogenicity of the labeled tumor was able to deter metastatic progression in a manner similar to that obtained by drug treatment. We conclude that the immunogenicity of xenobiotic labeling markers can significantly influence disease progression and therapeutic responses thereby compromising immunocompetent preclinical cancer models, and that the glowing-head mouse can be used to circumvent such problems and promote consistent disease monitoring. Citation Format: Chi-Ping Day, Zoe Weaver, John Carter, Carrie Bonomi, Terry Van Dyke, Melinda Hollingshead, Glenn Merlino. Immunological naturalization of immunocompetent host mice to luciferase-GFP for consistent tracking of transplanted tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2013-1556

Cre‐mediated recombination in pituitary somatotropes

We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick beta-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in. Cre activity is detectable in the developing pituitary after initiation of Gh transcription and persists through adulthood with high penetrance in Gh expressing cells and lower penetrance in lactotropes, a cell type that shares a common origin with somatotropes. This Gh-cre transgenic line is suitable for efficient, cell-specific deletion of floxed regions of genomic DNA in differentiated somatotropes and a subset of lactotrope cells of the anterior pituitary gland.

Epstein-Barr virus-based vector improves the tumor cell killing effect of pituitary tumor in HVJ-liposome-mediated transcriptional targeting suicide gene therapy

Although tissue-specific promoters offer a promising approach to the targeting of gene therapy, the activity of such promoters is generally low, which is thus a major limitation, especially when using non-viral vectors. To establish effective transcriptional targeting gene therapy for growth hormone (GH) producing pituitary tumors, an Epstein-Barr virus (EBV) based vector system expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) driven by a rat GH promoter (pEBGTK) was developed. This harbors an EBV nuclear antigen-1 (EBNA-1) gene with an origin of the latent viral DNA replication (OriP) gene of EBV. We constructed an EBV-based luciferase plasmid (pEBGL) as a reporter plasmid. We also generated pGTK and pGL, which are non-EBV counterparts. Metastatic GH3 (mGH3) cells were used in this study. The transfection of pEBGL to mGH3 resulted in approximately a 39 times greater luciferase activity than pGL in vitro. Its expression was also prolonged 144 h after transfection. According to the results of pEBGL gene transfer in in vivo experiments, the luciferase activity was only observed in the tumors, but not detected in other normal tissues. The luciferase activities in tumor tissues were found until day 25 post transfection. During in vitro gene therapy, the transfection by pEBGTK using hemmaglutinating virus of Japan (HVJ) liposome enhances the susceptibility of mGH3 to gancyclovir (GCV) 110 times more than that by pGTK. The in vivo anti-tumor effects of pEBGTK on mGH3-tumor-bearing nude mice were evaluated. The intratumoral injection of HVJ anionic lipososme-enveloped pEBGTK followed by the intra-peritoneal injection of GCV demonstrated a significant growth inhibition against tumors without toxicity, while the tumors treated by other treatment modalities grew progressively. These results demonstrated that the EBV-based vector system can therefore contribute to the improvement of the anti-tumor effects for the HVJ-liposome-mediated transcriptional targeting suicide gene therapy, suggesting that this paradigm may thus be a potentially effective approach for the treatment of uncontrollable pituitary tumors.

Functional Sequestration of Transcription Factor Activity by Repetitive DNA

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBPalpha. Functional sequestration of C/EBPalpha was demonstrated by experimentally reducing C/EBPalpha binding to the major alpha-satellite DNA, which elevated the concentration of C/EBPalpha in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBPalpha binding to alpha-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBPalpha from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBPalpha that reduces binding to alpha-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of alpha-satellite DNA binding coincided with an elevation in the binding of C/EBPalpha to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBPalpha to this highly repetitive DNA reduced the amount of C/EBPalpha available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

Binding of the Thyroid Hormone Receptor to a Negative Element in the Basal Growth Hormone Promoter Is Associated with Histone Acetylation

Nuclear thyroid hormone receptors (TRs) act as ligand-dependent activators, but paradoxically unliganded TRs can increase transcription of promoters containing negative response elements (nTRE), and hormone binding represses this activation. The rat growth hormone (GH) promoter contains a positive TRE and a nTRE. Ligand-dependent negative regulation mediated by the nTRE could play an important physiological role in restricting GH gene expression in non-pituitary cells that express TRs. With chromatin immunoprecipitation assays, we show here that the nTRE is responsible for binding of TR to the promoter in non-pituitary HeLa cells and that this element also governs transactivation by the unoccupied receptor and repression by triiodothyronine. Occupancy of the promoter by TR is concomitant with appearance of acetylated histone H3, and triiodothyronine causes release of the receptor as well as disappearance of the acetylated histone from the promoter. Although the nTRE overlaps the TATA box, the receptor does not exclude binding of TATA-binding protein, but could rather facilitate formation of the preinitiation complex. Furthermore, the proximal GH promoter is synergistically stimulated by unliganded TR and TATA-binding protein, whereas the ligand represses this cooperation. Constitutive receptor activity and synergism with TATA-binding protein require binding of corepressors. Furthermore, inhibitors of histone deacetylases enhance promoter activation by the unliganded receptor and reduce triiodothyronine-dependent repression, whereas expression of HDAC1 reverses promoter stimulation. This suggests that partitioning of histone acetylases and deacetylases between the receptors and basal transcription factors could be involved in regulation of the basal GH promoter by TRs.

CLONING OF THE FULL LENGTH PIG PIT1 (POU1F1) CDNA AND A NOVEL ALTERNATIVE PIT1 TRANSCRIPT, AND FUNCTIONAL STUDIES OF THEIR ENCODED PROTEINS

PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin β subunit (TSHβ). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3′ end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the Δ3PIT1 (missing entire exon 3), Δ4PIT1 (missing entire exon 4) and PIT1β (additional 26 amino acids inserted in front of exon 2) transcripts. The Δ4PIT1 and PIT1β transcripts have been found to encode functionally different proteins in rodents. The Δ3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig Δ3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and Δ3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig Δ3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between Δ3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that Δ3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel Δ3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.

Characterization of the Promoter-Directing Expression of Growth Hormone in a Monocyte Cell Line

Previous work from our laboratory has shown that cells of the immune system produce a growth hormone (GH) molecule similar to that produced by the pituitary. In the present study, using Southern analysis of RT-PCR products and sequencing of cloned cDNA molecules, we demonstrate that lymphoid cell lines utilize the same promoter and first exon as the pituitary somatotrope. To identify the cis-elements involved in transcriptional regulation of immune cell-derived GH, we have coupled rat GH promoter fragments to a luciferase reporter gene and transfected a monocyte cell line (P-388) by electroporation. The results suggest the presence of both positive (–299/–193 bp) and negative (–193/–107 bp) regulatory elements. The same constructs transfected in the pituitary cell line, GH3, in contrast to the monocyte cell line, showed a gradual decrease in luciferase expression. The overexpression of GHF-1 or GHF-2 resulted in a modest but significant reduction in rat GH promoter activity in the P-388 cell line. Taken together, the data suggest that immune cells utilize the same first exon and promoter sequence for the expression of monocyte GH as that reported for the expression of pituitary GH. Further, it appears that sequences between –299 and –107 bp are important in the regulation of the promoter where different transcription factors may be recruited to promote GH expression in a monocyte cell line.

Role of GHF-1 in the Regulation of the Rat Growth Hormone Gene Promoter by Thyroid Hormone and Retinoic Acid Receptors

In non-pituitary HeLa cells the unliganded thyroid hormone or retinoic acid receptors cause a strong activation of the rat growth hormone promoter that is repressed by their ligands. In contrast, after expression of the pituitary-specific transcription factor GHF-1, thyroid hormone and retinoic acid produce a stimulation similar to that found in pituitary cells. Therefore, GHF-1 changes a ligand-dependent inhibition into a ligand-dependent activation. The essential role of GHF-1 on the rat growth hormone promoter was also demonstrated with AF-2-defective T3 receptor mutants that show a normal activation of this promoter in the presence of GHF-1. Furthermore, a truncated T3 receptor, which lacks the N-terminus and the DNA binding domain, was able to stimulate this promoter in the presence of GHF-1 and exogenous RXR receptors, suggesting the importance of protein to protein interactions in this regulation. This study shows that the final transcriptional effect depends not only on the type of regulatory promoter response elements but also on the presence of other transcriptional activators, in the case of the growth hormone promoter, the tissue-specific transcription factor GHF-1, which plays a coactivator-like role in this promoter.

A direct protein-protein interaction is involved in the cooperation between thyroid hormone and retinoic acid receptors and the transcription factor GHF-1.

The nuclear receptors for thyroid hormone (TRs) and retinoic acid (RARs and RXRs) cooperate with the pituitary-specific transcription factor GHF-1 to activate the rat growth hormone (GH) gene. The GH promoter contains a hormone response element (HRE), which binds TR/RXR and RAR/RXR heterodimers, located close to two binding sites for GHF-1. GHF-1 inhibits binding of TR/RXR and RAR/RXR heterodimers to an isolated HRE. Similarly, the receptors inhibit binding of GHF-1 to its cognate site. These results suggest the existence of direct protein to protein interactions between the receptors and the pituitary transcription factor. This was confirmed by in vitro binding studies with GST fusion proteins, which demonstrated a strong association of GHF-1 with RXR and a weaker interaction with RAR and TR. GHF-1 and the receptor heterodimers form a ternary complex with a fragment of the rat GH promoter, which contains binding sites for both, and GHF-1 increases receptor binding to the promoter when present in limiting conditions. These results suggest that the synergistic activation of the rat GH gene involves protein-DNA interactions as well as a physical association between the nuclear receptors and the pituitary-specific transcription factor GHF-1.

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

Pituitary-directed leukemia inhibitory factor transgene forms Rathke's cleft cysts and impairs adult pituitary function. A model for human pituitary Rathke's cysts.

Leukemia inhibitory factor (LIF) and LIF receptors are expressed in adenohypophyseal cells and LIF regulates pituitary hormone transcription and cell replication in vitro. Therefore, transgenic mice expressing pituitary-directed LIF driven by the rat growth hormone (GH) promoter were generated to evaluate the impact of LIF on pituitary development. Three founders were established with diminished linear growth and body weight (57-65% of wild type [WT]), and intense anterior pituitary LIF immunoreactivity. Cystic cavities observed in pituitary anterior lobes were lined by cuboidal, ciliated epithelial cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones. Transgenic pituitaries showed decreased GH (40%) and prolactin (PRL) (26%) cells, and decreased GH and PRL mRNAs by in situ hybridization. ACTH cells increased 2.2-fold, whereas gonadotrophs and thyrotrophs were unchanged. Serum GH was undetectable (< 0.78 ng/ml), PRL levels were one third of WT (P < 0.05), IGF-I levels were 30% of WT (P < 0. 001), and T4 was normal. 10 human pituitary Rathke's cysts studied all showed conclusive LIF immunoreactivity in cyst-lining cells. Thus, intrapituitary murine LIF overexpression causes cystic invaginations from the anterior wall of Rathke's cleft, suggesting failed differentiation of Rathke's epithelium to hormone-secreting cells. Arrested murine pituitary maturation with formation of pituitary Rathke's cleft cysts, GH deficiency, and short stature provide a model to study human Rathke's cyst pathogenesis.

GHF-1/Pit-1 Functions as a Cell-specific Integrator of Ras Signaling by Targeting the Ras Pathway to a Composite Ets-1/GHF-1 Response Element

Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/GHF-1 binding site located between positions -217 and -190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes.

CCAAT/Enhancer-binding Protein α Activation of the Rat Growth Hormone Promoter in Pituitary Progenitor GHFT1-5 Cells

High level, anterior pituitary-specific expression of the rat growth hormone (rGH) promoter requires cooperative actions of several different transcription factors. Previously, we described a series of multisubunit, tissue-general, transcription factor complexes that bound to the GHF3 activation site and strongly regulated rGH promoter activity. A 43-kDa DNA-binding subunit common to each of the different GHF3 complexes is identified here as the transcription factor, CCAAT/Enhancer-binding Protein alpha (C/EBPalpha). In human monocyte U937 cells, which do not express the endogenous or transfected GH genes, co-expression of C/EBPalpha and Pit-1 synergistically activated the transfected rGH promoter. Full-length C/EBPalpha was present in the GH-secreting GC, and prolactin-secreting 235-1, pituitary cell lines, but not in GHFT1-5 cells, which are transformed at a stage in development immediately prior to GH expression. Transient expression of C/EBPalpha in GHFT1-5 cells strongly activated the co-transfected rGH promoter through the GHF3 binding site; a second activation site mapped to evolutionary conserved GH promoter sequences between -106 and -33. C/EBPalpha activation was synergistic with phorbol 12-myristate 13-acetate and forskolin, activators of protein kinases C and A, respectively. Thus, C/EBPalpha is an important regulator of rGH promoter activity that appears to function in synergy with Pit-1, activators of A and C protein kinases and possibly other factors.

An Activation Function in Pit-1 Required Selectively for Synergistic Transcription

Synergistic transcription activation is a key component in the generation of the spectrum of eukaryotic promoter activities by a limited number of transcription factors. Various mechanisms could account for synergy, but a central question remains of whether synergism requires transcription factor functions that differ from those that direct independent activation. The rat growth hormone promoter is synergistically activated by the pituitary-specific transcription factor, Pit-1, and the thyroid hormone receptor (TR). Mutations that disrupted the previously described DNA binding and transcriptional activation domains of both Pit-1 and TR reduced Pit-1/TR synergy in parallel with their effects on the much weaker, independent Pit-1 and TR activations of the rat growth hormone promoter. Thus, Pit-1 and TR amplify each other's intrinsic activities. Mutations of Pit-1 that selectively inhibited synergism with the TR without affecting independent Pit-1 activity were also identified. Pit-1/TR synergy is therefore a consequence of a novel synergism-selective activity and synergism-independent Pit-1 and TR functions.

Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Expression of constitutively active Gs alpha-subunits in GH3 pituitary cells stimulates prolactin promoter activity.

Although G protein alpha subunits are known to regulate such cellular functions as growth and enzymatic activity, the ability of these proteins to regulate target gene expression has not yet been directly investigated. Transient expression in GH3 pituitary cells of a target rat prolactin promoter-chloramphenicol acetyltransferase construct, (-1957)PRL-CAT, was increased by coexpressed constitutively active alpha s mutant Q227L-alpha s but not by wild-type alpha s. Thus activated alpha s but not basal state alpha s can stimulate prolactin promoter activity. Q227L-alpha s also stimulated expression of construct (-187)PRL-CAT, showing that only the proximal prolactin promoter region is required for a response to activated alpha s. The promoter specificity of the transcriptional influence of activated alpha s was demonstrated by the inability of either Q227L-alpha s or wild-type alpha s to stimulate expression of control target constructs containing either the rat growth hormone promoter or the thymidine kinase promoter. Previous studies have shown that the most proximal prolactin promoter binding site for the pituitary-specific transcription factor pit-1, site 1P, can act as an independent response element for either thyrotropin-releasing hormone or Ca2+. Two copies of site 1P conferred upon a heterologous metallothionein promoter a response to Q227L-alpha s. This implies that site 1P can also serve as an independent response element for alpha s and suggests that pit-1 may be a mediator of the cellular regulation by alpha s of the prolactin promoter.

In vitro transcriptional studies of the roles of the thyroid hormone (T3) response elements and minimal promoters in T3-stimulated gene transcription.

Thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that are encoded by two different genes, TR alpha and TR beta, and bind to thyroid hormone response elements (TREs) in the promoters of thyroid hormone (T3)-regulated genes. Retinoid X receptors (RXRs), major members of the thyroid hormone receptor auxiliary proteins, have recently been shown to enhance the binding of TRs to TREs. We previously showed that TRs extracted from rat pituitary GH3 cells retain ligand (T3) and DNA binding specificity and stimulate rat growth hormone (rGH) promoter activity in a cell-free in vitro transcription system. In this report, we have studied further how T3 activates endogenous TRs and stimulates transcription from different TRE-containing promoters. We found that T3 (10(-8) M) selectively stimulates transcription from rGH-TRE- and TREpal-, but not ME-TRE- and F2-TRE-, containing templates in which these TREs are linked in front of the rGH minimal promoter containing only the TATA box binding protein, but not any other proximal binding protein, sequence. In contrast, only the TREpal/AdML template, in which TREpal oligonucleotide was linked in front of the adenovirus major late gene (AdML) minimal promoter, was stimulated by T3. Electrophoretic mobility shift assay (EMSA) demonstrates that endogenous TR complexes specifically bind to either natural or idealized TRE (rGH-TRE, TREpal, ME-TRE, and F2-TRE) oligonucleotides. To further understand these receptor-DNA complexes formed on various TREs, isoform-specific anti-receptor antisera (TR alpha, TR beta 1, TR beta 2, and RXR beta) were added in the EMSA. These antisera differentially supershifted TR.DNA complexes formed on the TREs. These data suggest either that endogenous TR isoforms and RXR beta may form different complexes on the various TREs or that TR.RXR complexes have distinct conformations when bound to the various TREs. Taken together, these data suggest that particular TREs in which specific TR.RXR complexes are formed and different minimal promoters may provide specificity in T3-mediated transcriptional stimulation of gene expression.

Growth retardation in rats whose growth hormone gene expression was suppressed by antisense RNA transgene

We produced four transgenic founder rats (F0) by introducing into rat embryos a fusion gene, which consisted of rat growth hormone (GH) promoter containing with four copies of thyroid hormone response element (TRE) and antisense cDNA sequences for rat GH. This transgene promoter directed 2.8-fold stimulation of CAT gene expression in transfected GH3 rat pituitary tumor cells compared with the rat GH promoter alone. Two of four transgenic rats expressed antisense RNA in the pituitary. Transgenic offspring (F1) from each founder rat exhibited dwarfism at as early as 3-4 weeks of age, and they exhibited approximately 70-85% reduced growth rate compared with their nontransgenic littermates over 56 weeks of observation. Plasma rat GH concentration was approximately 40-50% lower in transgenic F1 rats compared to their nontransgenic littermates. In these experiments, the pituitary hormone expression controlled in a complex manner was shown to be repressed by the antisense RNA transgene. Furthermore, the suppression of gene expression could be achieved by antisense RNA transgene in the rat as well.

Distance-dependent interactions between basal, cyclic AMP, and thyroid hormone response elements in the rat growth hormone promoter

Developmental stage- and tissue-specific expression of the rat growth hormone (rGH) gene is conferred by DNA sequences within 237 base pairs of the transcription start site. Although binding of a number of transcription factors including Pit-1, Sp1, GHF3, and thyroid hormone receptor (T3R) stimulates rGH expression, several studies have suggested that interactions between these factors are important in determining cell specificity and responsiveness to extracellular signals. We have directly tested this hypothesis by creating a set of nested insertional mutations at two positions in the rGH promoter. Sequences were inserted at either position -148, separating GHF-3 and T3R binding sites from the downstream Pit-1 and Sp 1 binding sites, or at -51, separating the above elements from the TATA box. All insertions were made in the context of the rGH gene -237/+8 5'-flanking DNA, linked to a chloramphenicol acetyltransferase reporter gene and tested for activity by transient transfection in GC pituitary tumor cells. Insertions at both -148 and -51 caused sharp distance-dependent reductions in serum-stimulated expression such that insertions of 23 base pairs at -51 or 44 base pairs at -148 were sufficient to isolate the effects of sequences upstream of the insertion point. Insertions at -148 reduced T3 responsiveness severalfold but had little or no effect on stimulation by forskolin, whereas insertions at -51 reduced both T3 and forskolin responsiveness. Our results are consistent with the hypothesis that expression and regulation of the rGH gene is dependent on short-range protein-protein interactions, which are more critically dependent on spacing than the relative orientation of the transcription factor binding sites.

Altered Growth Hormone-Releasing Hormone mRNA Expression in Transgenic Mice with Excess or Deficient Endogenous Growth Hormone

Hypothalamic expression of growth hormone-releasing hormone (GHRH) mRNA was examined in two transgenic mouse models displaying excess or deficient endogenous GH. Transgenic dwarf mice bore a gene construct consisting of the rat growth hormone (GH) promoter fused to a diphtheria toxin A chain structural gene (DT-A); the GH promoter restricted DT-A expression to endogenous GH-producing cells, which were destroyed. GH was undetectable in either the pituitary or the peripheral circulation (Behringer et al., Genes Devel. 2: 453-461, 1988). Transgenic giant mice carried a construction joining the metallothionein promoter to the human GHRH structural gene, which stimulated endogenous pituitary GH production (Hammer et al., Nature 315: 413-417, 1985). In situ hybridization to GHRH mRNA in transgenic dwarf, giant, and nontransgenic controls was performed using single-stranded RNA probes generated from cloned mouse GHRH cDNA. Hybridization to GHRH mRNA was limited to the neurons of the hypothalamic arcuate nucleus (ARC). Autoradiographic densities on X-ray films were quantified by computerized image analysis. There was an increase in GHRH signal intensity in the dwarfs (282 ± 20 units; mean ± SEM) relative to that measured in control animals (107 ± 8 units; P < 0.001), while giant mice had decreased signal (42 ± 7 units; P < 0.001) in the ARC. The present studies demonstrate that increase in GHRH mRNA expression accompanies GH deficiency, while a decrease in GHRH mRNA accompanies GH excess, suggesting both positive and negative feedback upon steady-state mRNA levels in hypophysiotropic neurons by target pituitary hormone.