Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala=> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala=> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp muration, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG => AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH. (C) 2000 Academic Press.

Intermittent Fugu parathyroid hormone 1 (1–34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats

Human parathyroid hormone (hPTH) is currently the only treatment for osteoporosis that forms new bone. Previously we described a fish equivalent, Fugu parathyroid hormone 1 (fPth1) which has hPTH-like biological activity in vitro despite fPth1(1–34) sharing only 53% identity with hPTH(1–34). Here we demonstrate the in vivo actions of fPth1(1–34) on bone. In study 1, young male rats were injected intermittently for 30 days with fPth1 [30 μg–1000 μg/kg body weight (b.w.), (30fPth1–1000fPth1)] or hPTH [30 μg–100 μg/kg b.w. (30hPTH–100hPTH)]. In proximal tibiae at low doses, the fPth1 was positively correlated with trabecular bone volume/total volume (TbBV/TV) while hPTH increased TbBV/TV, trabecular thickness (TbTh) and trabecular number (TbN). 500fPth1 and 1000fPth1 increased TbBV/TV, TbTh, TbN, mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) with a concomitant decrease in osteoclast surface and number. In study 2 ovariectomized (OVX), osteopenic rats and sham operated (SHAM) rats were injected intermittently with 500 μg/kg b.w. of fPth1 (500fPth1) for 11 weeks. 500fPth1 treatment resulted in increased TbBV/TV (151%) and TbTh (96%) in the proximal tibiae due to increased bone formation as assessed by BFR/BS (490%) and MAR (131%). The effect was restoration of TbBV/TV to SHAM levels without any effect on bone resorption. 500fPth1 also increased TbBV/TV and TbTh in the vertebrae (L6) and cortical thickness in the mid-femora increasing bone strength at these sites. fPth1 was similarly effective in SHAM rats. Notwithstanding the low amino acid sequence homology with hPTH (1–34), we have clearly established the efficacy of fPth1 (1–34) as an anabolic bone agent.

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Melanocortin-1 receptor gene variants in four Chinese ethnic populations

There is strong relationship between melanocortin-1 receptor (MC1R) gene variants and human hair color and skin type. Based on a sequencing study of MC1R gene in 50 individuals from the Uygur, Tibetan, Wa and Dai ethnic populations, we discuss the occurre

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Expanding the spectrum of PTH1R mutations in patients with primary failure of tooth eruption

Objectives: Primary failure of tooth eruption (PFE) is a rare autosomal-dominant disease characterized by severe lateral open bite as a consequence of incomplete eruption of posterior teeth. Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been shown to cause PFE likely due to protein haploinsufficiency. To further expand on the mutational spectrum of PFE-associated mutations, we report here on the sequencing results of the PTH1R gene in 70 index PFE cases. Materials and methods: Sanger sequencing of the PTH1R coding exons and their immediate flanking intronic sequences was performed with DNA samples from 70 index PFE cases. Results: We identified a total of 30 unique variants, of which 12 were classified as pathogenic based on their deleterious consequences on PTH1R protein while 16 changes were characterized as unclassified variants with as yet unknown effects on disease pathology. The remaining two variants represent common polymorphisms. Conclusions: Our data significantly increase the number of presently known unique PFE-causing PTH1R mutations and provide a series of variants with unclear pathogenicity which will require further in vitro assaying to determine their effects on protein structure and function. Clinical relevance: Management of PTH1R-associated PFE is problematic, in particular when teeth are exposed to orthodontic force. Therefore, upon clinical suspicion of PFE, molecular DNA testing is indicated to support decision making for further treatment options. © 2013 Springer-Verlag Berlin Heidelberg.

Parathyroid hormone-related peptide (PTHrP) regulates fetal–placental calcium transport through a receptor distinct from the PTH/PTHrP receptor

To determine the role of PTHrP in fetal calcium metabolism, blood calcium was measured in mice homozygous (HOM) for deletion of the PTHrP gene. On day 18.5 of gestation, ionized calcium and the maternal–fetal calcium gradient were significantly reduced in HOM PTHrP-ablated fetuses compared with that of their littermates. To assess the placental contribution to the effect of PTHrP, 45Ca and 51Cr-EDTA (as a blood diffusional marker) were administered by intracardiac injection to pregnant, heterozygous dams on day 17.5 of gestation. Five minutes after the injection, whole fetal 45Ca accumulation was significantly decreased in HOM PTHrP-ablated fetuses compared with that of their littermates. Next, two fetuses from each litter were injected in utero with fragments of PTHrP, PTH, or diluent 1 h before administering 45Ca and 51Cr to the dam. PTHrP-(1–86) and PTHrP-(67–86) significantly increased relative 45Ca accumulation in HOM PTHrP-ablated fetuses, but PTHrP(1–34), PTH-(1–84), and the diluent had no effect. Finally, similar studies were performed on fetal mice that lacked the PTH/PTHrP receptor gene. Ionized calcium was significantly reduced in HOM PTH/PTHrP receptor-ablated fetuses. However, 5 min after maternal injection of 45Ca and 51Cr, relative accumulation of 45Ca was significantly increased in these fetuses. It was concluded that PTHrP is an important regulator of fetal blood calcium and placental calcium transport. In addition, the bioactivity of PTHrP for placental calcium transport is specified by a mid-molecular region that does not use the PTH/PTHrP receptor.

Identification of a long non-coding RNA gene, GHSROS (growth hormone secretagogue receptor opposite strand), which stimulates cell migration in non-small cell lung cancer cell lines

The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Morphologic changes and the expression of alpha-melanocyte stimulating hormone and melanocortin-1 receptor in melasma lesions: A comparative study

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light- to dark-brown macules on sun-exposed skin areas. The literature shows few studies on its physiopathogeny. However, changes in α-melanocyte stimulating hormone (α-MSH) secretion and melanocortin-1 receptor (MC1-R) expression may play a role to trigger this condition. Biopsies were taken from both melasma skin and adjacent perilesional normal skin of 44 patients. The biopsies were submitted for hematoxylin and eosin and Fontana-Masson staining and immunohistochemistry with Melan-A, α-MSH, and MC1-R, and processed for transmission electron microscopy. In some cases, they were submitted to MC1-R gene expression analysis by real-time polymerase chain reaction. Increased lymphohistiocytic infiltrate and solar elastosis, higher epidermal melanin were observed in melasma skin. Electron microscopy revealed a greater number of mature melanosomes in keratinocytes and melanocytes, and more prominent cytoplasmic organelles in melasma skin. There was no difference in melanocyte number between areas. However, melanocytes were larger and more dendritic in melasma skin. Immunohistochemistry with α-MSH and MC1-R showed significant labeling in melasmic epidermis but MC1-R messenger ribonucleic acid (RNAm) did not show significant quantitative difference between melasma and normal skin. © 2010 by Lippincott Williams & Wilkins.

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3)

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.

Tissue-specific response of the human platelet-activating factor receptor gene to retinoic acid and thyroid hormone by alternative promoter usage.

We have studied the effects of retinoic acid (RA) and thyroid hormone (3,3',5-triiodothyronine; T3) on platelet-activating factor receptor (PAFR) gene expression in intact rats and the ability of two human PAFR gene promoters (PAFR promoters 1 and 2) to generate two transcripts (PAFR transcripts 1 and 2). Northern blotting showed that RA and T3 regulated PAFR gene expression only in rat tissues that express PAFR transcript 2. Functional analysis of the human PAFR promoter 2 revealed that responsiveness to RA and T3 was conferred through a 24-bp element [PAFR-hormone response element (HRE) located from -67 to -44 bp of the transcription start site, whereas PAFR promoter 1 did not respond to these hormones. The PAFR-HRE is composed of three direct repeated TGACCT-like hexamer motifs with 2-and 4-bp spaces, and the two upstream and two downstream motifs were identified as response elements for RA and T3. Thus, the PAF-PAFR pathway is regulated by the PAFR level altered by a tissue-specific response to RA and T3 through the PAFR-HRE of the PAFR promoter 2.