Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus)

In the goldfish (Carassius auratus) the two endogenous forms of gonadotropin-releasing hormone (GnRH), namely chicken GnRH II ([His5,Trp7,Tyr8]GnRH) and salmon GnRH ([Trp7,Leu8]GnRH), stimulate the release of both gonadotropins and growth hormone from the pituitary. This control is thought to occur by means of the stimulation of distinct GnRH receptors. These receptors can be distinguished on the basis of differential gonadotropin and growth hormone releasing activities of naturally occurring GnRHs and GnRHs with variant amino acids in position 8. We have cloned the cDNAs of two GnRH receptors, GfA and GfB, from goldfish brain and pituitary. Although the receptors share 71% identity, there are marked differences in their ligand selectivity. Both receptors are expressed in the pituitary but are differentially expressed in the brain, ovary, and liver. Thus we have found and cloned two full-length cDNAs that appear to correspond to different forms of GnRH receptor, with distinct pharmacological characteristics and tissue distribution, in a single species.

Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium in vivo and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.

Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

Synthesis and biological activities of potent peptidomimetics selective for somatostatin receptor subtype 2

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 μg/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054,522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

Erythropoietin induces tumor regression and antitumor immune responses in murine myeloma models

Recombinant human erythropoietin (rHuEpo) has been used successfully in the treatment of cancer-related anemia. Clinical observations with several patients with multiple-myeloma treated with rHuEpo has shown, in addition to the improved quality of life, a longer survival than expected, considering the poor prognostic features of these patients. Based on these observations, we evaluated the potential biological effects of rHuEpo on the course of tumor progression by using murine myeloma models (MOPC-315-IgAλ2 and 5T33 MM-IgG2b). Here we report that daily treatment of MOPC-315 tumor-bearing mice with rHuEpo for several weeks induced complete tumor regression in 30–60% of mice. All regressors that were rechallenged with tumor cells rejected tumor growth, and this resistance was tumor specific. The Epo-triggered therapeutic effect was shown to be attributed to a T cell-mediated mechanism. Serum Ig analysis indicated a reduction in MOPC-315 λ light chain in regressor mice. Intradermal inoculation of 5T33 MM tumor cells followed by Epo treatment induced tumor regression in 60% of mice. The common clinical manifestation of myeloma bone disease in patients with multiple-myeloma was established in these myeloma models. Epo administration to these tumor-bearing mice markedly prolonged their survival and reduced mortality. Therefore, erythropoietin seems to act as an antitumor therapeutic agent in addition to its red blood cell-stimulating activity.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Organization and chromosomal localization of the gene encoding the mouse acid labile subunit of the insulin-like growth factor binding complex.

After birth, most of insulin-like growth factor I and II (IGFs) circulate as a ternary complex formed by the association of IGF binding protein 3-IGF complexes with a serum protein called acid-labile subunit (ALS). ALS retains the IGF binding protein-3-IGF complexes in the vascular compartment and extends the t1/2 of IGFs in the circulation. Synthesis of ALS occurs mainly in liver after birth and is stimulated by growth hormone. To study the basis for this regulation, we cloned and characterized the mouse ALS gene. Comparison of genomic and cDNA sequences indicated that the gene is composed of two exons separated by a 1126-bp intron. Exon 1 encodes the first 5 amino acids of the signal peptide and contributes the first nucleotide of codon 6. Exon 2 contributes the last 2 nt of codon 6 and encodes the remaining 17 amino acids of the signal peptide as well as the 580 amino acids of the mature protein. The polyadenylylation signal, ATTAAA, is located 241 bp from the termination codon. The cDNA and genomic DNA diverge 16 bp downstream from this signal. Transcription initiation was mapped to 11 sites over a 140-bp TATA-less region. The DNA fragment extending from nt -805 to -11 (ATG, +1) directed basal and growth hormone-regulated expression of a luciferase reporter plasmid in the rat liver cell line H4-II-E. Finally, the ALS gene was mapped to mouse chromosome 17 by fluorescence in situ hybridization.

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

A nuclear hormone receptor-associated protein that inhibits transactivation by the thyroid hormone and retinoic acid receptors.

Nuclear hormone receptors are transcription factors that require multiple protein-protein interactions to regulate the expression of their target genes. Using the yeast two-hybrid system, we identified a protein, thyroid hormone receptor uncoupling protein (TRUP), that specifically interacts with a region of the human thyroid hormone receptor (TR) consisting of the hinge region and the N-terminal portion of the ligand binding domain in a hormone-independent manner. Interestingly, TRUP inhibits transactivation by TR and the retinoic acid receptor but has no effect on the estrogen receptor or the retinoid X receptor in mammalian cells. We also demonstrate that TRUP exerts its action on TR and retinoic acid receptor by interfering with their abilities to interact with their DNA. TRUP represents a type of regulatory protein that modulates the transcriptional activity of a subclass of the nuclear hormone receptor superfamily by preventing interaction with their genomic response elements.

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.

Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.

Calcium-dependent homotypic cell-cell adhesion, mediated by molecules such as E-cadherin, guides the establishment of classical epithelial cell polarity and contributes to the control of migration, growth, and differentiation. These actions involve additional proteins, including alpha- and beta-catenin (or plakoglobin) and p120, as well as linkage to the cortical actin cytoskeleton. The molecular basis for these interactions and their hierarchy of interaction remain controversial. We demonstrate a direct interaction between F-actin and alpha (E)-catenin, an activity not shared by either the cytoplasmic domain of E-cadherin or beta-catenin. Sedimentation assays and direct visualization by transmission electron microscopy reveal that alpha 1(E)-catenin binds and bundles F-actin in vitro with micromolar affinity at a catenin/G-actin monomer ratio of approximately 1:7 (mol/mol). Recombinant human beta-catenin can simultaneously bind to the alpha-catenin/actin complex but does not bind actin directly. Recombinant fragments encompassing the amino-terminal 228 residues of alpha 1(E)-catenin or the carboxyl-terminal 447 residues individually bind actin in cosedimentation assays with reduced affinity compared with the full-length protein, and neither fragment bundles actin. Except for similarities to vinculin, neither region contains sequences homologous to established actin-binding proteins. Collectively these data indicate that alpha 1 (E)-catenin is a novel actin-binding and -bundling protein and support a model in which alpha 1(E)-catenin is responsible for organizing and tethering actin filaments at the zones of E-cadherin-mediated cell-cell contact.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.