bluuebunny/crossref_metadata_2025 huggingface dataset:
Repression of the acetyl-CoA carboxylase gene in ovine adipose tissue during lactation: the role of insulin responsiveness
Authors: Travers MT , Vernon RG , Barber MC
Published: December 2004

We have investigated the mechanisms whereby lipogenesis is markedly suppressed in adipose tissue depots of lactating sheep. Expression of the gene encoding acetyl-CoA carboxylase (ACC), the flux-determining enzyme of the lipogenic pathway, is reduced approximately threefold in both omental and subcutaneous adipose tissue depots during late pregnancy and remains so into lactation when compared with non-pregnant, non-lactating animals. By comparison, total ACC enzyme activity in these adipose depots is suppressed approximately 25- to 30-fold in lactation. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an approximately sevenfold increase in ACC mRNA, a fivefold increase in total enzyme activity and a marked increase in the proportion of the enzyme in the active state when compared with explants cultured with no added hormones for the same period. However, there was a lag of between 32 and 48 h before marked induction of any of these parameters by insulin plus dexamethasone was observed. Induction of the alpha-tubulin gene paralleled that of the ACC gene, suggesting that cytoskeletal rearrangements are associated with the aquisition of sensitivity to insulin plus dexamethasone. These results demonstrate that the reduction in lipogenic capacity in ovine adipose tissue during lactation is related to repression of the ACC gene, both at the level of steady-state mRNA abundance and possibly at translation, as well as to suppression of the mechanisms that regulate the proportion of ACC in the active state, and these are further related to the marked insensitivity of these parameters to insulin plus dexamethasone in vitro.
The regulation of mammary prolactin receptor metabolism by a retroviral envelope protein
Authors:   Bolander FF, Ginsburg E , Vonderhaar BK
Published: December 2004

In a previous study, infection with the mouse mammary tumor virus (MMTV) was shown to increase the sensitivity of the mammary epithelium toward prolactin (PRL); furthermore, this effect could be mimicked by the binding of the MMTV envelope protein (gp52) to its cell receptor. The present work has investigated the possibility that gp52-induced changes in the PRL receptor (PRLR) were responsible for this phenomenon. In vitro, gp52 doubled the PRLR concentration in the plasmalemma of mammary epithelium without affecting the affinity. The origins of these PRLRs were twofold: first, gp52 stimulated PRLR mRNA nearly fivefold, suggesting that some of the receptors were newly synthesized. Second, there was a redistribution of PRLRs within the mammary cell: PRLRs were shifted from an internal pool to the plasma membrane. This relocation was very rapid, occurring within 30 min. There did not appear to be any contribution from alterations in PRLR degradation, since the half-life of PRLR was not affected by gp52. In summary, the MMTV increases the PRL sensitivity of mouse mammary epithelium by elevating PRLRs through both enhanced synthesis and recruitment from microsomes.
Distinct promoter sequences of two precursor genes for salmon gonadotropin-releasing hormone in masu salmon
Authors: Higa M , Kitahashi T , Sasaki Y , Okada H , Ando H
Published: December 2004

Two types of genes encode salmon gonadotropin-releasing hormone (sGnRH), which is thought to act on both sexual maturation and reproductive behavior, in salmonids. We characterized the two sGnRH genes (sGnRH-I and -II) and their upstream regions in masu salmon, Oncorhynchus masou, since such information is a prerequisite for molecular approaches to salmon reproduction. The two genes have similar exon-intron structures composed of four exons and three introns. Sequence analyses of the two genes showed that coding regions are highly conserved, but upstream regions are distinctively divergent. In the upstream regions, only the sGnRH-II gene has a large palindromic sequence, which has been proposed to be involved in control of transcription via estrogen receptors. In contrast, the sGnRH-I gene is missing the large palindromic sequence, but has three distinct palindromes in the upstream region. These results may suggest divergent transcription regulatory mechanisms between the two sGnRH genes in masu salmon. The differences in the upstream regions of sGnRH genes in Atlantic salmon (Salmo salar), sockeye salmon (Oncorhynchus nerka) and masu salmon are discussed with respect to the evolution of sGnRH genes in salmonid fish.
Expression of neurotrophin-3 in the growing velvet antler of the red deer Cervus elaphus
Authors: Garcia RL , Sadighi M , Francis SM , Suttie JM , Fleming JS
Published: December 2004

Antlers are organs of bone which regenerate each year from the heads of male deer. In addition to bone, support tissues such as nerves also regenerate. Nerves must grow at up to 1 cm/day. The control of this rapid growth of nerves is unknown. We examined the relative expression of neurotrophin-3 (NT-3) mRNA in the different tissues of the growing antler tip and along the epidermal/dermal layer of the antler shaft of the red deer Cervus elaphus, using semi-quantitative reverse transcription-polymerase chain reaction. Expression in the tip was found to be highest in the epidermal/dermal layer and lowest in the cartilaginous layer in all developmental stages examined. These data correlate well with the density and pattern of innervation of these tissues. Along the epidermal/dermal layer of the antler shaft, expression was highest in the segments subjacent to the tip and lowest near the base, arguing for differences in the temporal expression of NT-3 in these segments. The expression of NT-3 in cells isolated from the different layers of 60-day antlers did not mirror that observed when whole tissues were used and may suggest regional specificity of NT-3 expression within antler tissues.
Alternate splicing of the RNA for hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4isomerase
Authors: Rogerson FM , LeHoux JG , Lefebre A , Mason JI
Published: December 2004

Complementary DNAs encoding the hamster type 2 3 beta-hydroxysteroid dehydrogenase/delta 5-->4 isomerase were isolated from liver and kidney cDNA libraries. Nine clones were isolated containing identical coding and 3' untranslated sequences. However, six of the clones contained a 68-nucleotide stretch in the 5' untranslated region that was missing in the other three clones. Primers were designed to flank this region and the polymerase chain reaction (PCR) was performed on hamster liver and adrenal RNA. Two PCR products were amplified of the predicted molecular sizes and with the expected sequence. Primers were then designed to amplify sequences encompassing this region from hamster genomic DNA. Sequencing of the resultant PCR products demonstrated that the 68-nucleotide stretch missing in some transcripts corresponded exactly to the second of three exons identified. We conclude that the 5' untranslated region of this mRNA is transcribed from at least three exons, and that the sequence of the second of these exons is spliced out of some of the RNA transcripts.
Cloning and characterisation of the gene encoding red deer (Cervus elaphus) growth hormone: implications for the molecular evolution of growth hormone in artiodactyls
Authors: Lioupis A , Wallis OC , Wallis M
Published: December 2004

In mammals the structure of pituitary GH is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and of artiodactyls, the rate of evolution has increased dramatically (25- to 50-fold) so that the sequences of human and ruminant GHs differ markedly from those of other mammalian GHs. In order to define further the burst of GH evolution that occurred in artiodactyls we have cloned and characterised the GH gene of red deer (Cervus elaphus) using genomic DNA and a polymerase chain reaction technique. The deduced sequence for the mature GH from red deer is identical to that of bovine GH, indicating that the burst of rapid evolution of GH that occurred in Artiodactyla must have been completed before the divergence of Cervidae and Bovidae and suggesting that the rate of evolution during this burst must have been greater than previously estimated. In other aspects (signal sequence, 5' and 3' sequences, introns and synonymous substitutions in the coding sequence) the red deer GH gene differs considerably from the GH genes of other ruminants. Differences between the signal peptide sequences of red deer and bovid GHs probably explain why N-terminal heterogeneity is seen in bovine, ovine and caprine GHs but not GH from red deer, pig or most other mammals.
Cloning and characterization of the rat gene for the acid-labile subunit of the insulin-like growth factor binding protein complex
Authors: Delhanty PJ , Baxter RC
Published: December 2004

The acid-labile subunit (ALS) of the ternary insulin-like growth factor-binding protein complex has a central role in controlling the bioavailability of circulating insulin-like growth factors. We have shown that gene expression of ALS is regulated by a number of factors, particularly growth hormone. Our aim was to characterize the ALS gene in order to define the mechanism of its regulation. Southern analysis suggests a single copy of the ALS gene in the rat genome. The protein-coding and 3'-untranslated regions span approximately 3.5 kilobases of rat genome and are divided into two exons. The 5' flanking region of the gene lacks a consensus TATA-box or Inr sequence, and primer extension and reverse transcriptase PCR experiments locate multiple transcriptional initiation sites between -505 and -385 nucleotides relative to the translational initiation codon. This putative promoter region, when inserted upstream of the luciferase reporter gene, directs luciferase expression when transfected into H4-II-E cells. Our data demonstrate the uncomplicated structure of the rat ALS gene, and the promoter function and presence of potential regulatory elements in the region upstream of the protein-coding sequence.
The mouse estrogen receptor-related orphan receptor alpha 1: molecular cloning and estrogen responsiveness
Authors: Shigeta H , Zuo W , Yang N , DiAugustine R , Teng CT
Published: December 2004

Estrogen receptor-related orphan receptor alpha 1 is a member of the steroid/thyroid nuclear receptor superfamily. We have previously cloned the human estrogen receptor-related orphan receptor alpha 1 (hERR alpha 1) cDNA and demonstrated that it enhances estrogen responsiveness of the lactoferrin gene promoter in transfected human endometrial carcinoma cells. In the present study, we used the hERR alpha 1 cDNA as a probe and isolated the mouse homologue of ERR alpha 1 from the cDNA libraries of the brain and kidney. Sequence comparison between human and mouse ERR alpha 1 (mERR alpha 1) revealed that the homologies are 89% in nucleotides and 97% in amino acids. By electrophoresis mobility shift assay, we showed that the glutathione S-transferase-mERR alpha 1 fusion protein produced in a bacterial system bound to the human ERR alpha 1 DNA-binding element. Mouse uterine nuclear extract also interacted with this DNA element and produced three complexes in the mobility shift assay, one of which was supershifted by the hERR alpha 1 antiserum. A 2.2 kbp transcript was detected by Northern analysis in all adult mouse tissues tested; however, large variations in the amount of ERR alpha 1 mRNA were found among them. Multiple immunoreactive forms of mouse ERR alpha 1 were detected by Western analysis in non-reproductive tissues, whereas a major 53 kDa protein was found in reproductive tissues such as uterus, cervix and vagina. Diethylstilbestrol (DES) stimulated the expression of ERR alpha 1 mRNA in the uterus of 19-day-old mouse. We showed that DES and estradiol, but not progesterone or dexamethasone, enhanced the level of immunoreactive ERR alpha 1 in the mouse uterus. These results demonstrated that the ERR alpha 1 is an estrogen-responsive gene in the mouse uterus and provides a model system with which to study the biological roles of this nuclear orphan receptor.
Two complementary bioassays for screening the estrogenic potency of xenobiotics: recombinant yeast for trout estrogen receptor and trout hepatocyte cultures
Authors: Petit F , Le Goff P , Cravedi JP , Valotaire Y , Pakdel F
Published: December 2004

A relation between the chemical structure of a xenobiotic and its steroidal action has not yet been clearly established. Thus, it is not possible to define the estrogenic potency of different xenobiotics. An assessment may be accomplished by the use of different bioassays. We have previously developed a yeast system highly and stably expressing rainbow trout estrogen receptor (rtER) in order to analyze the biological activity of the receptor. The recombinant yeast system appears to be a reliable, rapid and sensitive bioassay for the screening and determination of the direct interaction between ER and estrogenic compounds. This system was used in parallel with a more elaborate biological system, trout hepatocyte aggregate cultures, to examine the estrogenic potency of a wide spectrum of chemicals commonly found in the environment. In hepatocyte cultures, the vitellogenin gene whose expression is principally dependent upon estradiol was used as a biomarker. Moreover, competitive binding assays were performed to determine direct interaction between rtER and xenobiotics. In our study, 50% of the 49 chemical compounds tested exhibited estrogenic activity in the two bioassays: the herbicide diclofop-methyl; the fungicides biphenyl, dodemorph, and triadimefon; the insecticides lindane, methyl parathion, chlordecone, dieldrin, and endosulfan; polychlorinated biphenyl mixtures; the plasticizers or detergents alkylphenols and phthalates; and phytoestrogens. To investigate further biphenyl estrogenic activity, its principal metabolites were also tested in both bioassays. Among these estrogenic compounds, 70% were able to activate rtER in yeast and hepatocytes with variable induction levels according to the system. Nevertheless, 30% of these estrogenic compounds exhibited estrogenic activity in only one of the bioassays, suggesting the implication of metabolites or different pathways in the activation of gene transcription. This paper shows that it is important to combine in vivo bioassays with in vitro approaches to elucidate the mechanism of xenoestrogen actions.
Expression and purification of biologically active porcine follicle-stimulating hormone in insect cells bearing a baculovirus vector
Authors: Kato Y , Sato I , Ihara T , Tomizawa K , Mori J , Geshi M , Nagai T , Okuda K , Kato T , Ueda S
Published: December 2004

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.
Mouse 17 beta-hydroxysteroid dehydrogenase type 2 mRNA is predominantly expressed in hepatocytes and in surface epithelial cells of the gastrointestinal and urinary tracts
Authors: Mustonen MV , Poutanen MH , Kellokumpu S , de Launoit Y , Isomaa VV , Vihko RK , Vihko PT
Published: December 2004

17 beta-Hydroxysteroid dehydrogenase (17HSD) type 2 efficiently catalyzes the conversion of the high activity 17 beta-hydroxy forms of sex steroids into less potent 17-ketosteroids. In the present study in situ hybridization was utilized to analyze the cellular localization of 17HSD type 2 expression in adult male and female mice. The data indicate that 17HSD type 2 mRNA is expressed in several epithelial cell layers, including both absorptive and secretory epithelia as well as protective epithelium. In both males and females, strong expression of 17HSD type 2 was particularly detected in epithelial cells of the gastrointestinal and urinary tracts. The mRNA was expressed in the stratified squamous epithelium of the esophagus, and surface epithelial cells of the stomach, small intestine and colon. The hepatocytes of the liver and the thick limbs of the loops of Henle in the kidneys, as well as the epithelium of the urinary bladder, also showed strong expression of 17HSD type 2 mRNA in both male and female mice. In the genital tracts, low 17HSD type 2 expression was detected in the seminiferous tubules, the uterine epithelial cells and the surface epithelium of the ovary. Expression of the mRNA was also detected in the sebaceous glands of the skin. The results indicate that in both male and female mice, 17HSD type 2 is expressed mainly in the various epithelial cell types of the gastrointestinal and urinary tracts, and therefore suggest a role for the enzyme in steroid inactivation in a range of tissues and cell types not considered as classical sex steroid target tissues.
The effect of the peroxisome proliferator ciprofibrate on the gastric mucosa and particularly the gastrin cell
Authors: Waldum HL , Kvetnoi IM , Sylte R , Schulze B , Martinsen TC , Sandvik AK
Published: December 2004

The peroxisome proliferator ciprofibrate induces hypergastrinemia without inhibiting acid secretion. The present study was carried out to assess the effect of ciprofibrate on serum gastrin and gastrin (G) cells in different strains of rats and to compare the effect of ciprofibrate with other lipid-reducing agents (lovastatin and simvastatin) which have a different mechanism of action. Serum gastrin was determined by a radioimmunoassay method, G cell density by histomorphometry after immunostaining for G cells, and gastrin, somatostatin and histidine decarboxylase (HDC) mRNA abundance by Northern blot analysis. Ciprofibrate (100 mg/kg/day for three weeks) induced a marked hypergastrinemia (P < 0.01) in male and female Fischer rats as well as in female Wistar rats. Simvastatin and lovastatin did not affect serum gastrin. Antral G cell density increased significantly in female Wistar rats (P < 0.05) and non-significantly in the other rats after ciprofibrate. Both gastrin and somatostatin mRNA abundance in antral mucosa increased markedly and significantly (P < 0.01) after ciprofibrate treatment. The present study shows that the peroxisome proliferator ciprofibrate induces hypergastrinemia secondary to an increased storage and synthesis of antral gastrin. Since somatostatin mRNA abundance also increased, the present study suggests that ciprofibrate and possibly other peroxisome proliferators in sufficient concentrations have a stimulatory effect on endocrine cells.
The analysis of regulatory sequences required for high level induction of rat alpha 2u-globulin gene expression by dexamethasone
Authors: Wang KS , Hodgetts RB
Published: December 2004

The alpha 2u-globulins are the major urinary proteins in adult male rats. They are encoded by a gene family, the expression of which is under multihormonal control in the liver. Glucocorticoids are positive regulatory hormones and we have analyzed the contribution of 5'-upstream sequences to the induction by dexamethasone of two cloned members of the family transfected into mouse L-cells. The results demonstrate that sequences from -762 bp to -226 bp of clone 91 are required for the 24-fold level of induction that was observed. Addition of 5.5 kb of upstream sequence beyond -762 bp did not alter the level of induction significantly, whereas deletion of the DNA between -762 bp and -226 bp reduced inducibility to about 4-fold. Sequencing of this region revealed that an element, 5'-AGAACAggtTTCAAA-3', similar to the 15 bp consensus glucocorticoid response element 5'-AGAACAnnnTGTACC-3', is situated 513 bp upstream of the transcription start site. We infer that this element or its left half site is necessary for the dexamethasone-induced expression of clone 91 from the observation that a second gene, clone 2, that contained a base substitution at position 5 in the left half site was not inducible. It now appears that at least three distinct cis-acting regulatory regions, all of which bind to the glucocorticoid receptor in vitro, may contribute to the full induction of clone 91 by dexamethasone. These are: the distal upstream region identified by this study, a proximal upstream region that binds not only the receptor but also alpha 2uNF1, a constitutively expressed nuclear protein required for induction and a region within the fourth intron that contains five tandem receptor binding sites.
Suppression subtractive hybridization (SSH) identifies prolactin stimulation of p38 MAP kinase gene expression in Nb2 T lymphoma cells: molecular cloning of rat p38 MAP kinase
Authors: Nemeth E , Bole-Feysot C , Tashima LS
Published: December 2004

Suppression Subtractive Hybridization (SSH) has been used to compare rat Nb2 cells treated with prolactin for 1 hour with untreated cells. This new method for identifying differentially expressed genes showed that the mRNAs for at least three genes were elevated by such treatment, including a p38 mitogen activated protein (MAP) kinase. The p38 MAP kinase was cloned and the full length cDNA sequence was determined.
Growth inhibition of both MCF-7 and Hs578T human breast cancer cell lines by vitamin D analogues is associated with increased expression of insulin-like growth factor binding protein-3
Authors: Colston KW , Perks CM , Xie SP , Holly JM
Published: December 2004

The effects of two vitamin D analogues, EB1089 and CB1093, on insulin-like growth factor binding protein (IGFBP) expression have been examined in MCF-7 and Hs578T human breast cancer cell lines. Both vitamin D analogues inhibited IGF-1 stimulated growth of MCF-7 cells and enhanced the production of IGFBP-3 as determined by Western-ligand blotting. Recombinant human IGFBP-3 inhibited the growth of MCF-7 cells over the concentration range 1-235 ng/ml. Hs578T cells were unresponsive to the mitogenic effects of IGF-1 but growth was inhibited by the two vitamin D analogues. Treatment of Hs578T cells with EB1089 and CB1093 (10 nM) as well as 100 nM 9-cis retinoic acid (9-cis RA) or all-trans retinoic acid (ATRA) was associated with increased accumulation of IGFBP-3 in conditioned medium. Furthermore, cotreatment of Hs578T cells with EB1089 and 9-cis RA led to augmented effects on both inhibition of cell growth and IGFBP-3 accumulation in conditioned medium as assessed by Western ligand blotting and radioimmunoassay. These findings suggest a role for IGFBP-3 in the growth inhibitory effects of vitamin D analogues.
Is prolactin a gonadotrophic hormone in red deer (Cervus elaphus)? Pattern of expression of the prolactin receptor gene in the testis and epididymis
Authors: Jabbour HN , Clarke LA , McNeilly AS , Edery M , Kelly PA
Published: December 2004

This study investigated the pattern and site of expression of the prolactin receptor gene in the testis and epididymis of red deer collected during the breeding season (n=3). Ribonuclease protection assays using 50 microg total RNA and a 300 bp [32P]-labelled antisense cRNA probe, generated from the extracellular domain of the red deer prolactin receptor, confirmed the expression of the receptor in both the testis and epididymis; a higher level of prolactin receptor mRNA was detected in the epididymis compared with the testis (170.4+/-1.5 x 10(3) and 26.3+/-2.7 x 10(3) arbitrary units respectively; P<0.05). In situ hybridisation using 300 bp [33P]-labelled sense and antisense cRNA probes generated from the extracellular domain of the receptor localised the expression sites to the seminiferous tubules and interstitial compartments of the testis and the epithelial layer of the epididymal duct. Quantification of grain numbers demonstrated a higher level of expression of the receptor in the epididymis compared with the interstitial and seminiferous tubule compartments of the testis (18.1+/-4.4 x 10(2), 10.1+/-2.0 x 10(2) and 8.3+/-0.8 x 10(2) grains/microm2 respectively; P<0.05). However, no differences were detected in the level of expression of the receptor between the interstitial and seminiferous tubule compartments of the testis. Immunocytochemistry using an anti-prolactin receptor antibody, raised against a peptide sequence from the extracellular domain of the rat prolactin receptor, localised expression of the receptor gene to the Leydig cells, pachytene spermatocytes, round spermatids and elongating spermatids. In the epididymis, the receptor was localised to the epithelial layer within the epididymal ducts. Expression of the prolactin receptor gene in the red deer testis and epididymis suggests a role for the hormone in steroidogenesis and spermatogenesis.
Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization
Authors: Huang WH , Lau AT , Daniels LL , Fujii H , Seydel U , Wood DJ , Papadimitriou JM , Zheng MH
Published: December 2004

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.
Tumour necrosis factor-alpha stimulates increased expression of prostaglandin endoperoxide H synthase Type 2 mRNA in amnion-derived WISH cells
Authors: Hansen WR , Sato T , Mitchell MD
Published: December 2004

We have evaluated the mechanism by which tumour necrosis factor-alpha (TNF-alpha) induces increased prostaglandin (PG) biosynthesis in amnion-derived WISH cells. WISH cells were treated with 50 ng/ml TNF-alpha or vehicle for 0-24 h. PGE2 production was stimulated by TNF-alpha within 2 h and continued to accumulate for at least 24 h. Increased prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression was evident within 30 min and was highest by 1 h, returning to unstimulated levels by 2 h. The PGHS-2 mRNA was re-induced at 8 h and was also elevated at 16 h. Immunoreactive PGHS-2 protein was nearly undetectable in control cells. However, within 30 min of TNF-alpha treatment, PGHS-2 protein was elevated and was induced for at least 16 h suggesting rapid production of both the PGHS-2 mRNA and protein. Transcription run-on assays indicated that the initial increase in the PGHS-2 mRNA was due to a 20-fold increase in the rate of transcription. The PGHS-2 mRNA decayed with an apparent half-life of 1 h in TNF-alpha-stimulated WISH cells. Induction of PGHS-2 expression proceeded in the presence of 10 microg/ml cycloheximide which agrees with the classification of PGHS-2 as an immediate early gene. These results indicate that a bi-phasic induction of the PGHS-2 mRNA is due, in part, to an initial transcriptional activation which results in rapid and continued synthesis of the PGHS-2 protein. This may be a unique characteristic of amnion cells which may be partially responsible for increased PG concentrations in the amniotic fluid during infection-associated preterm labour.
Modulation of mouse estrogen receptor transcription activity by protein kinase C delta
Authors: Lahooti H , Thorsen T , Aakvaag A
Published: December 2004

The effect of protein kinase C (PKC) delta on the transcriptional activity of the mouse estrogen receptor was investigated. The receptor was expressed transiently in Cos-1 and NIH3T3 cells in the presence of wild-type, dominant negative or constitutively active forms of PKC delta. Transfection experiments demonstrated that PKC delta stimulated both unliganded and liganded estrogen receptor transcriptional activity. This stimulatory effect was not observed using PKC alpha or PKC epsilon. 4-Hydroxytamoxifen and the pure anti-estrogen ICI 164,384 reduced receptor transcriptional activity in the presence of PKC delta. The stimulatory effect of PKC delta on estrogen receptor transcriptional activity was mediated by the N-terminal activation function 1 (AF-1) domain. The reduced stimulatory effect of PKC delta on transcriptional activity of the phosphorylation defective mutant of estrogen receptor suggests that phosphorylation of serine 122 in the AF-1 region may mediate the modulatory effect of PKC delta. Wild-type PKC delta caused a twofold increase in estrogen receptor phosphorylation, while a dominant negative mutant of PKC delta reduced the receptor phosphorylation to five percent of that caused by wild-type PKC delta. Our results suggest that PKC delta participates in the signaling pathways that lead to estrogen receptor phosphorylation and its effect on estrogen receptor transcriptional activation is both cell type and promoter specific.
Beta2-adrenoceptor desensitization in non-pregnant estrogen-primed rat myometrium involves modulation of oxytocin receptor gene expression
Authors: Engstrom T , Bratholm P , Vilhardt H , Christensen NJ
Published: December 2004

The nona-peptide oxytocin (OT) induces contraction of the myometrium by interaction with specific plasma membrane associated OT receptors (OTR), whereas stimulation of beta2-adrenoceptors (beta2AR) causes relaxation. Homologous desensitization of the myometrium to both hormones has been described. However, a possible interaction between the two systems has not been investigated. In the present study, long-term in vivo treatment of non-pregnant estrogen-primed rats with isoproterenol decreased maximal relaxation of isolated uterine strips challenged with isoproterenol. Increased EC50 values of similarly treated animals suggest that the coupling between receptor occupancy and contractile response was impaired. Since beta2AR mRNA levels were left unchanged, we conclude that the homologous desensitization to beta2 stimulation is not due to changes in beta2AR gene expression. OT infusion did not alter beta2AR mRNA levels or isoproterenol-induced relaxation of isolated uterine strips. Treatment with OT had no effect on the amount of myometrial OTR mRNA. We have previously found that OT down-regulates OTR in the non-pregnant rat myometrium, but this therefore does not appear to take place at the level of mRNA production. Isoproterenol treatment resulted in a three-fold increase in OTR mRNA. This was accompanied by a 91% rise in OTR binding and an augmented contractile response of isolated uterine strips to OT, suggesting that the increased production of mRNA reflects formation of active receptors. Neither OTR affinity nor EC50 of in vitro strips was affected by isoproterenol treatment. We conclude that stimulation of beta2AR causes heterologous up-regulation of OTR in the non-pregnant estrogen-primed rat myometrium.
Cloning of bovine parathyroid hormone-related protein (PTHrP) cDNA and expression of PTHrP mRNA in the bovine mammary gland
Authors: Wojcik SF , Schanbacher FL , McCauley LK , Zhou H , Kartsogiannis V , Capen CC , Rosol TJ
Published: December 2004

Parathyroid hormone-related protein (PTHrP) produced by the mammary gland has been postulated to have multiple functions in both the mother and neonate. In humans, alternative 3'-mRNA splicing and endoproteolytic processing result in multiple bioactive PTHrP peptides. Multiple PTHrP peptides also have been reported in bovine milk. To investigate the source of molecular heterogeneity of PTHrP in bovine milk, bovine PTHrP was cloned from a bovine brain cDNA library, sequenced and used to characterize the mammary PTHrP transcript. A 1065 bp clone (bP1) for bovine PTHrP was isolated from a brain cDNA library. The bP1 clone contained the entire coding sequence of PTHrP and 61 and 473 nucleotides of the 5'- and 3'-untranslated regions (UTRs) respectively. The predicted amino acid sequence of bovine PTHrP was 72-92% homologous to the sequences of chicken, rat, mouse, human, and canine PTHrP with the highest sequence divergence present in the C-terminal region of the peptide. The 5'- and 3'-UTRs of bovine brain PTHrP have a high degree of homology to exons 4 and 9 of human PTHrP respectively. PTHrP was expressed as a single 1200 nucleotide mRNA transcript in lactating bovine mammary tissue. RT-PCR using region-specific oligonucleotide primers derived from bP1 demonstrated that PTHrP mRNA transcripts in bovine brain and lactating mammary gland utilize the same 5'- and 3'-UTRs. Expression of PTHrP mRNA was localized to secretory and ductular epithelial cells within the lactating mammary gland, as detected using in situ hybridization. Expression of PTHrP mRNA was demonstrated in the mammary gland during late pregnancy and throughout lactation in cows.
Establishment of a stable steroidogenic porcine granulosa cell line
Authors: Chedrese PJ , Rodway MR , Swan CL , Gillio-Meina C
Published: December 2004

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 microM androstenedione elevated the levels of estradiol-17beta to 0.028, 0.3 and 1.21 pg/microg protein, respectively. The level of expression of 3betaHSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.
Characterization of angiotensin II receptors (binding and mRNA) in the rat thyroid gland
Authors: Montiel M , Jimenez E
Published: December 2004

In this study we showed, for the first time, the existence of a moderate density of specific angiotensin II (Ang II) binding sites (Kd=3.9+/-1.7 nM and Bmax=467.2 130.0 fmol/mg protein) in plasma membrane preparations from rat thyroid gland. Reverse transcriptase/polymerase chain reactions, using primers based on the cloned AT1 and AT2 receptor subtypes, and pharmacological characterization, using the Ang II receptor subtype antagonists Losartan and PD 123319, revealed that these Ang II binding sites match with the AT1 receptor subtypes. To obtain more information on the molecular structure of this Ang II receptor, immunoblotting analyses were carried out using a polyclonal rabbit anti-AT1 antiserum. Western analysis of fresh plasma membrane preparations from thyroid tissue showed three prominent bands of approximately 60, 45 and 40 kDa which appear to be related to different degrees of glycosylation of the receptor molecule. The functional significance of the Ang II receptors in thyroid gland is currently not known. Nevertheless, since Ang II receptors play a pivotal role in the co-ordinated actions of the renin-angiotensin system (RAS), our findings support a reciprocal regulation of thyroid function by the RAS.
Regulation of epidermal growth factor expression in mammary epithelial cells by a Yin-Yang-1-like element
Authors: Fang Y , Sheffield LG
Published: December 2004

The hormone combination of insulin, dexamethasone and prolactin induced accumulation of preproepidermal growth factor (EGF) mRNA in HC11 mouse mammary epithelial cells 16-24 h after the hormones were added to the cultures. Individual hormones or combinations of two of the hormones had no effect on EGF mRNA concentrations. The same hormone combination was capable of inducing expression of a reporter gene construct containing -888 to +25 bp of the EGF gene fused with luciferase. Deletions of the promoter between -888 and -271 bp had no detectable effect on basal or hormone-induced reporter gene expression. However, further deletion from -270 to -74 bp increased baseline to approximately equal hormone-induced reporter gene expression. This deletion also abolished the hormone-induced increase in reporter gene expression. Sequence analysis suggested that this region contained a binding site for Yin-Yang-1 (YY1), which was confirmed by gelshift analysis. Mutation of the YY1 binding site increased baseline reporter gene expression to the same level as induced by insulin, dexamethasone and prolactin in the wild-type promoter. These results indicate that expression of the EGF gene in mammary epithelium is repressed by the YY1 site, and that removal of repression may play a part in regulating EGF gene expression in lactating mammary tissue.
Characterization of the phosphatidylinositol-specific phospholipase C isozymes present in the bovine parathyroid and in human kidney HEK293 cells stably transfected with the human parathyroid Ca2+-sensing receptor
Authors: Dare E , Kifor O , Brown EM , Weber G
Published: December 2004

The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.
Glucocorticoid receptor polymorphism in genetic hypertension
Authors: Kenyon CJ , Panarelli M , Zagato L , Torielli L , Heeley RP , Holloway CD , Fraser R , Casari G , Sutcliffe RG , Bianchi G
Published: December 2004

The Milan hypertensive strain of rat (MHS) displays abnormalities in both renal function and adrenocortical activity. While the pressor role of the former has been studied in detail, the role of the latter has not yet been clearly evaluated. In the present study, glucocorticoid receptor (GR) binding characteristics in liver cytosol from adult MHS and Milan normotensive controls (MNS) have been investigated. Dexamethasone, aldosterone and corticosterone were bound with lower affinity to cytosol of MHS rats compared with that of MNS rats. This pattern of binding could explain the raised plasma corticosterone concentrations and adrenocortical hypertrophy previously noted in MHS. The coding sequence of MHS and MNS GR genes have been determined. The MHS gene differed in four respects from that of MNS: three silent point mutations and a polymorphic microsatellite region in exon 2. The latter polymorphism has been used in cosegregation studies of F2 hybrids of MHS x MNS. The MHS GR genotype was associated with hypercalciuria and lower blood pressure in female rats and lower body weight in male rats. Although the effect on blood pressure is small, it is consistent with the affinity data. MHS GR genotype cosegregated with lower blood pressure in F2 rats and displayed a lower affinity in binding studies. In conclusion, GR polymorphism may be responsible for differences of adrenocortical function between MHS and MNS. This may lead to a reduction in the blood pressure difference between the two strains.
Fetal rat lung epithelium has a functional growth hormone receptor coupled to tyrosine kinase activity and insulin-like growth factor binding protein-2 production
Authors: Batchelor DC , Lewis RM , Breier BH , Gluckman PD , Skinner SJ
Published: December 2004

Although growth hormone (GH) receptor (GHR) mRNA and protein are present in fetal tissues such as the lung, there is little evidence that GH mediates growth in the fetus. We have identified functional responses to GH in fetal rat lung epithelia and suggest a possible role for GHR in the developing lung. GHR mRNA in lung extracts was high before birth at day 16 of gestation (16f), decreased to low levels at day 22f but increased again after birth. At day 20f GHR mRNA levels were higher in lung than in liver, whereas growth hormone binding protein mRNA levels were approximately equal in lung and liver. Stimulation of primary cell cultures of day 19f lung epithelia with GH caused increased tyrosine phosphorylation in specific proteins, demonstrating functional GHR. Lung fibroblasts isolated at the same time did not respond to GH. Ligand and Northern blot analysis of the epithelial cultures revealed that GH stimulation increased insulin-like growth factor binding protein-2 (IGFBP-2) activity and mRNA. These experiments demonstrate the functional activity of GHR, specifically in fetal lung epithelium. We suggest that one role for GH in vivo may be indirectly to modify insulin-like growth factor activity in the developing fetal lung by increasing IGFBP-2.
Transcriptional activities in the pituitaries of channel catfish before and after induced ovulation by injection of carp pituitary extract as revealed by expressed sequence tag analysis
Authors: Karsi A , Li P , Dunham RA , Liu ZJ
Published: December 2004

Expressed sequence tag (EST) analysis was adopted to address physiological changes after injection of carp pituitary extract for induction of ovulation. ESTs were analyzed from cDNA libraries constructed from mRNA isolated from channel catfish (Ictalurus punctatus) pituitaries before and after induction of ovulation by injection of carp pituitary extract. One hundred randomly picked clones were analyzed. Of the sequences generated, a large percentage (59%) of ESTs were identified as known genes by identity comparisons. These 59 clones of known gene products represent transcriptional products of 30 genes. The 41 clones of unknown gene products represent 33 genes. Expression of gonadotropin (GtH) alpha-subunit (149%) and prolactin (176%) was slightly enhanced as a result of induced ovulation. Large increases in frequencies of several peptide hormones were observed as a result of induced ovulation: GtH beta-I, 486%; GtH beta-II, 933%; growth hormone, 393%; proopiomelanocortin (POMC), 345%. POMC represented about 21% of all transcriptional activity in the pituitaries after induced ovulation. This is the first study addressing physiological changes after injection of carp pituitary extract, a procedure widely used in catfish hatcheries.
Regulation of mammary hormone receptor metabolism by a retroviral envelope protein
Authors:   Bolander FF
Published: December 2004

In a previous study, the envelope protein (gp52) of the mouse mammary tumour virus (MMTV) was shown to facilitate mammary gland differentiation by increasing prolactin (PRL) receptors via increased receptor synthesis and via the redistribution of existing receptors from an internal pool. In this study, receptors for other hormones known to affect mammary gland metabolism were investigated. Epidermal growth factor (EGF) stimulates mammary epithelial growth and inhibits differentiation; its receptor is rapidly and dramatically down-regulated by gp52. This is accomplished by its internalization and by decreasing its half-life from 27 h to 2.4 h. Surprisingly, it also increased EGF receptor synthesis, although this effect was not great enough to overcome receptor down-regulation. In contrast, gp52 did not affect the distribution, half-life or synthesis of the insulin receptor. These results demonstrate that MMTV can enhance mammary differentiation by coordinately regulating several hormone receptors: specifically, it can increase the number of receptors for PRL, a differentiative hormone, while decreasing the number of receptors for EGF, a growth/anti-differentiative hormone.