bluuebunny/crossref_metadata_2025 huggingface dataset:
A new mutation of the androgen receptor, P817A, causing partial androgen insensitivity syndrome: in vitro and structural analysis
Authors: Lumbroso S , Wagschal A , Bourguet W , Georget V , Mazen I , Servant N , Audran F , Sultan C , Auzou G
Published: December 2004

Androgen insensitivity syndrome (AIS) is an X-linked disease caused by mutations in the androgen receptor (AR) resulting in various degrees of defective masculinization in 46,XY individuals. In the present study, we describe a novel mutation in exon 7 of the AR gene in an Egyptian patient with partial AIS (PAIS). Sequencing analysis of the AR gene revealed a novel missense mutation, P817A, within the ligand-binding domain (LBD). This is the first report of a mutation within the short amino acid motif (codons 815-817) of the beta-strand lying between helices H8 and H9 of the AR LBD. The functional defects of the mutated protein were characterized by in vitro study and included significantly decreased ligand-binding affinity and impaired transactivation potential. Limited proteolysis assays performed with the wild-type and mutant AR receptors incubated with the synthetic agonist R1881 revealed that the P817A mutation resulted in a reduced stabilization of the AR active conformation. Structural analyses showed that this mutation is likely to perturb the beta-sheet interaction between residues 815-817 and 911-913. This structural alteration destabilizes the position of the C-terminal extension, which contains residues critical for androgen function.
Reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in adrenal glomerulosa cells
Authors: Foster RH
Published: December 2004

The main regulators of aldosterone secretion in adrenal gland zona glomerulosa (ZG) cells are the hormones angiotensin II (Ang II) and adrenocorticotrophin (ACTH) and small increases in the extracellular potassium (K(+)) concentration. The action of these agonists is mediated by different signalling systems - ACTH is mediated by cAMP and activation of protein kinase A while Ang II and K(+) activate two protein kinases, Ca(2+)-calmodulin-dependent protein kinase (CamK) and diacylglycerol-dependent protein kinase (PKC). Ang II, besides being one of the main agonists for the secretion of aldosterone, also stimulates proliferation of ZG cells, a process mediated by mitogen-activated protein kinases (MAPKs). Recent studies aimed at elucidating the molecular mechanisms underlying cell proliferation have shown that calcineurin is the principal regulator of MAPKs activity. The purpose of this review is to discuss experimental evidence of possible reciprocal influences between the signalling pathways regulating proliferation and steroidogenesis in ZG cells.
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and hepatocyte nuclear factor 4gamma (HNF-4gamma) and HNF-4alpha regulate the bovine growth hormone receptor 1A promoter through a common DNA element
Authors: Xu Q , Walther N , Jiang H
Published: December 2004

The growth hormone receptor (GHR) 1A promoter is responsible for transcription of the liver-specific GHR mRNA variant 1A in several mammalian species. We previously found that the region between nucleotide -218 and nucleotide -151 (relative to the major transcription start site) of the bovine GHR 1A promoter contained a binding site between -196 and -178 for the liver-enriched transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) and that the same region might also interact with additional transcription factors in the liver. Using the -218/-151 region as bait to screen a bovine liver cDNA library in a yeast one-hybrid analysis, we identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and HNF-4gamma, as well as HNF-4alpha, as binding proteins to the -218/-151 region. These binding proteins were also identified in a second yeast one-hybrid analysis using the -196/-178 region as bait, suggesting that COUP-TFII, HNF-4gamma and HNF-4alpha all bind to the -196/-178 region. Electrophoretic mobility shift assays confirmed the ability of these three transcription factors in bovine liver nuclear extracts to interact with the -196/-178 region. These interactions also appeared to exist in vivo, as the GHR 1A promoter containing the -196/-178 region could be recovered by immunoprecipitation of the bovine liver chromatin with antibody against COUP-TFII, HNF-4gamma or HNF-4alpha. In co-transfection analyses, each of these three transcription factors could activate GHR 1A promoter and the activation was dependent on the -196/-178 region. These results together identify COUP-TFII, HNF-4gamma and HNF-4alpha as transcription factors regulating GHR 1A promoter activity through binding to a common DNA element.
Distinct effects of the antiestrogen Faslodex on the stability of estrogen receptors-alpha and -beta in the breast cancer cell line MCF-7
Authors: Peekhaus NT , Chang T , Hayes EC , Wilkinson HA , Mitra SW , Schaeffer JM , Rohrer SP
Published: December 2004

The effects of estrogen receptor (ER) ligands on the stability and transcriptional activity of ERbeta in the breast cancer cell lines MCF-7 and HeLa were examined. We found that ERbeta was degraded in the presence of 17beta-estradiol. Tamoxifen and Faslodex (ICI 182,780) prevented ERbeta receptor destabilization. In contrast to ERalpha, ERbeta degradation was not abolished by inhibitors of the proteasome-mediated protein degradation pathway. Furthermore, single point mutations in helix 12 of the receptor dramatically affected the stability and subsequent transcriptional activation of ERbeta.
Thyroid hormone regulation of prohormone convertase 1 (PC1): regional expression in rat brain and in vitro characterization of negative thyroid hormone response elements
Authors: Shen X , Li QL , Brent GA , Friedman TC
Published: December 2004

Most pro-neuropeptides are processed by the prohormone convertases, PC1 and PC2. We previously reported that changes in thyroid status altered anterior pituitary PC1 mRNA and this regulation was due to triiodothyronine (T(3))-dependent interaction of thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large region of the human PC1 promoter. In this study, we demonstrated that hypothyroidism stimulated, while hyperthyroidism suppressed, PC1 mRNA levels in rat hypothalamus and cerebral cortex, but not in hippocampus. In situ hybridization was used to confirm real-time PCR changes and localize the regulation within the hypothalamus and cortex. Using a human PC1 (hPC1) promoter construct (with and without deletions in two regions that each contain a negative TRE) transiently transfected into GH3 cells, we found that T(3) negatively regulated hPC1 promoter activity, and this regulation required both of these two regions. Electrophoretic mobility shift assays (EMSAs) using purified thyroid hormone receptor alpha1 (TRalpha1) and retinoid X receptor beta (RXRbeta) proteins demonstrated that RXR and TRalpha both bound the PC1 promoter. Addition of TRalpha1/RXRbeta to the wild-type PC1 probe demonstrated binding as both homodimers and a heterodimer. EMSAs with oligonucleotides containing deletion mutations of the putative nTREs demonstrated that the proximal nTRE binds more strongly to TR and RXR than the distal nTRE, but that both regions exhibit specific binding. We conclude that there are multiple novel TRE-like sequences in the hPC1 promoter and that these regions act in a unique manner to facilitate the negative effect of thyroid hormone on PC1.
Expression of two progesterone receptor isoforms in cumulus cells and their roles during meiotic resumption of porcine oocytes
Authors: Shimada M , Yamashita Y , Ito J , Okazaki T , Kawahata K , Nishibori M
Published: December 2004

The present study aimed to investigate progesterone receptor (PR) gene expression in cumulus cells and their roles during meiotic resumption of porcine oocytes. The amount of PR-A or PR-B mRNA was analyzed by RT-PCR using primer sets for the PR-B region or the PR-A/B common region. The level of PR-B mRNA in cumulus cells was up-regulated by FSH and LH during the first 8 h of cultivation but the level significantly decreased at 12 h. However, a high level of total PR mRNA was maintained up to a cultivation period of 20 h. The level of PR-B protein in cumulus cells reached its maximum at 4 to 12 h, whereas PR-A predominated in cumulus cells of cumulus-oocyte complexes (COCs) at 20 h. Accompanying the shift in expression of PR isoforms, progesterone production in cumulus cells was significantly increased, and both the proliferative activity of cumulus cells during a 10- to 20-h cultivation period and the level of connexin-43, a major component of the gap junction, in cumulus cells significantly decreased. When COCs were cultured with FSH and LH for 10 h and then further cultured with additional RU486, there was a significant suppression in the shift in PR isoforms and in progesterone production, a loss of proliferative activity, and a decrease in connexin-43 mRNA in cumulus cells. Moreover, treatment with RU486 after 10-h cultivation of COCs inhibited the meiotic resumption of oocytes and cumulus cell expansion. These results suggest that the induction of PR isoforms in cumulus cells and their binding to progesterone appear to impact on proliferation and differentiation in a time-dependent manner, and the shift from PR-B to PR-A may help mediate certain events.
Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression
Authors: Bolduc C , Larose M , Yoshioka M , Ye P , Belleau P , Labrie C , Morissette J , Raymond V , Labrie F , St-Amand J
Published: December 2004

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50 000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPα, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.
cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-α in NCI-H295R adrenocortical cells
Authors: Liu J , Li X-D , Ora A , Heikkilä P , Vaheri A , Voutilainen R
Published: December 2004

Adrenocorticotropin is the major regulator of adrenocortical development and function. It acts mainly through the cAMP-dependent protein kinase A (PKA) pathway. Our aim was to study the interaction of tumor necrosis factor-α (TNFα) and the PKA pathway in adrenocortical cell proliferation and apoptosis. The PKA activator Dibutyryl cAMP ((Bu)2cAMP) strongly induced differentiation and inhibited proliferation in the human adrenocortical cell line NCI-H295R (H295R). TNFα induced apoptosis of H295R cells. Interestingly, (Bu)2cAMP treatment clearly enhanced TNFα-induced apoptosis in H295R cells, but not in another human adrenocortical cell line SW-13, the mouse adrenocortical Y-1 cell line or the human HeLa cell line. This synergistic effect was not due to the (Bu)2cAMP-induced glucocorticoid secretion since dexamethasone had no significant effect on the TNFα-induced apoptosis. (Bu)2cAMP treatment rapidly increased the expression of the proto-oncogene c-myc in H295R cells, but not in SW-13, Y-1 or HeLa cells. In transient c-myc transfection assay, c-myc expression associated with decreased expression of the proliferation marker Ki-67 in H295R cells. In conclusion, cAMP-dependent protein kinase activation reduced proliferation and augmented TNFα-induced apoptosis in adrenocortical H295R cells, and these effects were associated with increased c-myc expression.
Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors α and β by coactivators and corepressors
Authors: Klinge C M , Jernigan S C , Mattingly K A , Risinger K E , Zhang J
Published: December 2004

One mechanism by which ligand-activated estrogen receptors α and β (ERα and ERβ) stimulate gene transcription is through direct ER interaction with specific DNA sequences, estrogen response elements (EREs). ERE-bound ER recruits coactivators that stimulate gene transcription. Binding of ER to natural and synthetic EREs with different nucleotide sequences alters ER binding affinity, conformation, and transcriptional activity, indicating that the ERE sequence is an allosteric effector of ER action. Here we tested the hypothesis that alterations in ER conformation induced by binding to different ERE sequences modulates ER interaction with coactivators and corepressors. CHO-K1 cells transfected with ERα or ERβ show ERE sequence-dependent differences in the functional interaction of ERα and ERβ with coactivators steroid receptor coativator 1 (SRC-1), SRC-2 (glucocorticoid receptor interacting protein 1 (GRIP1)), SRC-3 amplified in breast cancer 1 (AIB1) and ACTR, cyclic AMP binding protein (CBP), and steroid receptor RNA activator (SRA), corepressors nuclear receptor co-repressor (NCoR) and silencing mediator for retinoid and thyroid hormone recpetors (SMRT), and secondary coactivators coactivator associated arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 1 (PRMT1). We note both ligand-independent as well estradiol- and 4-hydroxytamoxifen-dependent differences in ER-coregulator activity. In vitro ER-ERE binding assays using receptor interaction domains of these coregulators failed to recapitulate the cell-based results, substantiating the importance of the full-length proteins in regulating ER activity. These data demonstrated that the ERE sequence impacts estradiol-and 4-hydroxytamoxifen-occupied ERα and ERβ interaction with coregulators as measured by transcriptional activity in mammalian cells.
Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding
Authors: Headey Stephen J , Leeding Kerri S , Norton Raymond S , Bach Leon A
Published: December 2004

Insulin-like growth factors IGF-I and IGF -II are important mediators of growth. A family of six high affinity IGF binding proteins (IGFBPs) modulate IGF action. IGFBPs have three domains, of which the N- and C-domains are involved in high affinity IGF binding. IGFBP-6 is unique in its 20–100-fold IGF-II binding specificity over IGF-I. The aim of this study was to determine the contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties. We confirmed that differential dissociation kinetics are responsible for the IGF-II binding preference of IGFBP-6. The N-domain has rapid association kinetics, similar to full-length IGFBP-6, but both IGF-I and -II dissociate rapidly from this domain, thereby reducing its binding affinity for IGF-II ~50-fold. However, the N-domain binds IGF-I and -II with similar affinities and it has a similar IGF-I binding affinity to full-length IGFBP-6. This suggests that the C-domain confers the IGF-II binding preference of IGFBP-6; indeed, IGF-I bound inconsistently with very low affinity to the C-domain. Coincubation studies showed that isolated N- and C-domains of IGFBP-6 do not strongly cooperate to enhance IGF binding. The results of the binding studies are supported by the effects of the IGFBP-6 domains on IGF-induced colon cancer cell proliferation; the N-domain inhibited IGF-II induced proliferation with ~20-fold lower potency than IGFBP-6 and it was equipotent in inhibiting IGF-I- and IGF-II-induced proliferation. Coincubation of C-domain had no additional effect on N-domain-induced inhibition of proliferation. In conclusion, both the N- and C-domains of IGFBP-6 are involved in IGF binding, the C-domain is responsible for the IGF-II binding preference of IGFBP-6 and intact IGFBP-6 is necessary for high affinity IGF binding.
Total amount of circulating human chorionic gonadotrophin α and β subunits in first trimester trisomies 21 and 18
Authors: Jauniaux E , Nicolaides K H , Nagy A-M , Brizot M , Meuris S
Published: December 2004

The aim of this study was to evaluate the variations in the balance between total (free and combined) circulating α and β subunits of human chorionic gonadotrophin (hCG) in trisomy 21 and 18.

Maternal serum samples were collected at 10 and 11 weeks of gestation from 22 singleton pregnancies with trisomy 21 (n=17) and trisomy 18 (n=5) and 66 chromosomally normal controls, matched for gestational age. The hCG and free α and β subunits circulating levels were measured using specific immunoradiometric assays and converted in a common unit system obtained using calibration of the assays with intact and thermally dissociated purified hCG preparation.

In trisomy 21, the only significant difference from controls was in the free βhCG level which was increased. In trisomy 18, intact hCG, free βhCG as well as total αhCG and total βhCG levels were significantly lower whereas the free αhCG level was significantly higher than in controls. The decrease in total βhCG was more pronounced than the decrease in total αhCG resulting in a significant increase in the total α- to βhCG subunit ratio in trisomy 18.

These findings suggest some modifications in the biosynthesis and/or release rates of the hCG subunits in these trisomies.

Journal of Endocrinology (1996) 148, 27–31
Thyroid hormone modulates androgen and oestrogen receptor content in the Sertoli cells of peripubertal rats
Authors: Panno M L , Sisci D , Salerno M , Lanzino M , Pezzi V , Morrone E G , Mauro L , Palmero S , Fugassa E , Andò S
Published: December 2004

A possible role of tri-iodothyronine (T3) on the interplay between testicular steroids and Sertoli cells has been investigated on the basis of previous findings demonstrating a direct inhibitory influence of T3 on aromatase activity and oestradiol production in peripuberal Sertoli cells. In this context, the present study was focused on the effects of T3 on oestrogen receptor (ER) and androgen receptor (AR) contents in the cytosol and nucleus of Sertoli cells isolated from 2-, 3- and 4-week-old euthyroid, hypothyroid and hypothyroid treated rats.

Hypothyroidism was induced by the oral administration of 0·025% methimazole (MMI) from birth until the rats were killed at 2, 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with l-tri-iodothyronine (T3; 3 μg/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Euthyroid ERs showed an elevated Kd (0·76 nm) which was similar in the different age groups investigated. The in vitro addition of T3 or testosterone induced a decrease in ER content and this decrease was greater after exposure to both hormones. In 2- and 3-week-old hypothyroid rats, ER content was markedly increased and was reversed in euthyroid rats when T3 was given in vivo. When ERs were assayed in the Sertoli cell nucleus and cytoplasm of 2- and 3-week-old animals, a strong relationship in ER content in the two cellular compartments was observed. Neither of the hormones tested seemed to affect the AR content in the nucleus significantly, while the in vitro addition of testosterone or T3 or both hormones together augmented the ARs in the cytosol to a greater extent, resulting in an increase in their total (cytosolic and nuclear) content in the cells.

The present data suggest that T3 down-regulates ERs and up-regulates ARs in peripuberal Sertoli cells. The additive effect of testosterone and T3 in up-regulating ARs could possibly involve a role for T3 in influencing the androgen responsiveness of the Sertoli cells during spermatogenesis.

Journal of Endocrinology (1996) 148, 43–50
Plasma levels and molecular forms of proatrial natriuretic peptides in healthy subjects and in patients with congestive heart failure
Authors: Azizi C , Maistre G , Kalotka H , Isnard R , Barthélemy C , Masson F , Pham P , Pousset F , Eurin J , Lechat P , Komajda M , Carayon A
Published: December 2004

A specific and sensitive radioimmunoassay (RIA) for the N-terminal fragment of proatrial natriuretic peptide (NproANP) was developed. Antiserum raised in rabbits against a mixture enriched with prohormone was 100% cross-reactive with human proANP(1–30). Plasma concentrations of proANP(1–30) and ANP immunoreactivities (ir-) were simultaneously measured in healthy subjects and patients with congestive heart failure (CHF; 26 dilated cardiomyopathy and 5 ischemic heart disease). High plasma levels of both ir-proANP(1–30) and ir-ANP were detected in CHF patients. Circulating ir-ANP levels were elevated in New York Heart Association functional Classes II and III patients but not in Class I patients. However, plasma levels of ir-proANP(1–30) were higher in asymptomatic patients than in healthy subjects, and markedly increased in patients of Classes II and III. Analysis of ir-proANP(1–30) by gel filtration chromatography or reverse-phase high pressure liquid chromatography revealed a 10 kDa peptide circulating as a distinct entity.

These findings indicate that: (i) the most probable form of NproANP in human plasma is a 10 kDa peptide and (ii) in CHF patients the rise in plasma ir-proANP(1–30) levels is more pronounced than the variation in plasma ir-ANP. Thus, NproANP plasma levels may prove to be a more sensitive marker of left ventricular dysfunction than ANP.

Journal of Endocrinology (1996) 148, 51–57
Leukaemia inhibitory factor in human endometrium throughout the menstrual cycle
Authors: Vogiagis D , Marsh M M , Fry R C , Salamonsen L A
Published: December 2004

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine previously demonstrated to be essential for blastocyst implantation in mice. Samples of endometrium from normal cyclic women throughout the menstrual cycle were tested for LIF messenger RNA by Northern blot analysis and the corresponding protein was localised immunohistochemically with a polyclonal antibody to LIF. Western blot analysis detected a 45 kDa LIF protein in an extract from late secretory tissue. The expression of LIF messenger RNA transcript was detected only during the mid and late secretory phases of the cycle after day 20. Immunoreactive LIF was observed in all human endometrial samples. In the stroma there were moderate to high levels of immunohistochemical staining throughout the cycle with considerable variation between individuals but no cyclical variation. Epithelial staining, both luminal and glandular, was also present throughout the cycle but this was relatively low in the proliferative phase and strongest in the mid to late secretory phases. The marked cyclical changes of immunoreactive LIF in the human endometrial epithelium suggest a paracrine/autocrine role for LIF in endometrial function. Whether LIF is essential for implantation in the human remains to be established.

Journal of Endocrinology (1996) 148, 95–102
Inhibitory effect of increased photoperiod on wool follicle growth
Authors: Pearson A J , Parry A L , Ashby M G , Choy V J , Wildermoth J E , Craven A J
Published: December 2004

The relationships between circulating prolactin (PRL), wool follicle growth and daylength were investigated in 24 New Zealand Wiltshire ewes housed indoors from September 1989 to May 1991. Twelve control (C) ewes were maintained under natural photoperiod. Two other groups were held in short days (SD; 8 h light:16 h darkness) commencing from the winter solstice (22 June 1990) for either three (group SD3, n=7) or six (group SD6, n=5) months before reversion to natural daylength. Skin was sampled at one- to four-week intervals for histological determination of percentages of growing primary and secondary follicles. Hourly blood samples over 24 h were collected via jugular cannulae from C sheep in March and July and then monthly from all animals until December 1990 for estimation of mean monthly PRL concentrations for each treatment group. Between autumn (March 1990) and winter (July) primary follicle activity (PFA) and secondary follicle activity (SFA) declined in C ewes (PFA: 97 to 43%, SFA: 100 to 57%). Follicle regrowth during July and August in eight C ewes preceded the initial rise in plasma PRL from the winter minimum (1·6 ng/ml). Across the three groups, four instances of decreased follicle activity were observed, closely following or concurrent with increases in plasma PRL concentrations. The resumption of spring growth in four C sheep was temporarily checked by falls in follicle activities during September and October as PRL concentrations began to increase (3·4 to 8·9 ng/ml). Follicle activity also declined in November and December in eight C sheep, coincident with the rapid rise in PRL to a seasonal maximum in late November (165·4 ng/ml). The increase in SD3 follicle activity over spring was not delayed by short days but during October, after release from treatment, PRL concentrations rose (1·8 to 12·0 ng/ml) and follicle activity declined (PFA: 65 to 38%, SFA: 68 to 43%). In SD6 ewes, PRL concentrations were suppressed (2·1 ng/ml) and relatively constant levels of follicle activity (PFA: 73%, SFA: 95%) were maintained throughout short-day treatment. Release of SD6 ewes into summer photoperiod in January 1991 temporarily interrupted follicle growth (PFA: 68 to 17%, SFA: 96 to 19%) and caused out-ofseason shedding in March and April. Contemporary C follicle activities were high (PFA: 95%, SFA: 98%). These data suggest that natural and experimental increases in daylength have a short-term inhibitory effect on growing wool follicles which could be mediated through rising concentrations of plasma prolactin.

Journal of Endocrinology (1996) 148, 157–166
17β-Oestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiation-dependent manner
Authors: Rao L G , Wylie J N , Kung Sutherland M S , Murray T M
Published: December 2004

We tested the effect of osteoblastic differentiation on the interactive effects of 17β-oestradiol (E2) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on alkaline phosphatase activity. As cell models we utilized the more differentiated human osteosarcoma (SaOS) cells that had been cultured for 6 days in medium containing 10 nm dexamethasone (Dex) (SaOS+Dex cells) and the less differentiated cells cultured in the absence of Dex (SaOS−Dex cells). The cells were challenged with 1,25(OH)2D3 in the presence or absence of Dex for 24 h and then with E2 for an additional 24 h. In SaOS−Dex cells, alkaline phosphatase activity remained constant over the 48-h period and was not significantly affected by E2, 1,25(OH)2D3 or 1,25(OH)2D3+E2 treatment. On the other hand, in SaOS+Dex cells, 1,25(OH)2D3 and E2+1,25(OH)2D3 stimulated alkaline phosphatase activity (ANOVA, F= 154·2, P<0·0001) with the maximal response at 48 h (P<0·01). In SaOS+Dex cells, 1,25(OH)2D3 had dose-dependent stimulatory effects which were strongly enhanced by 10 nm E2 (ANOVA, F=46·0, P<0·001). Studies on dose-dependent effects of E2, in the presence or absence of 100 nm 1,25(OH)2D3, revealed that in the presence of 1,25(OH)2D3, the E2 dose-response curve was biphasic in SaOS+Dex cells (ANOVA, F=3·40, P<0·005), with maximum stimulation at 10 nm E2 (P<0·01). The specificity of E2 was verified using the inactive 17α-oestradiol and the oestrogen antagonist, tamoxifen. These data indicate that E2 and 1,25(OH)2D3 have positive interactive effects on alkaline phosphatase activity in human osteoblasts, and suggest that the expression of this interaction is dependent on the stage of differentiation of the cells.

Journal of Endocrinology (1996) 148, 181–187
Characterisation of acyltransferase activity in ovine placental tissue during pregnancy and at the time of labour
Authors: Wong M H , Rice G E
Published: December 2004

Although it is well established that the formation of eicosanoids by ovine intrauterine tissues increases during pregnancy and at the time of labour, the biochemical mechanisms involved remain to be clearly established. In this study, we tested the hypothesis that the gestational and labour-associated increases in eicosanoid formation are associated with a reduction in the activity of the reacylating enzyme, acyl Coenzyme A lysophosphatide acyltransferase (LAT). To evaluate this proposal, in vitro LAT activity was quantified in ovine placenta (cotyledons) obtained during pregnancy (85–147 days of gestation and at the time of labour). Ovine placental LAT increased from 1·81 ± 0·06 nmol/min per mg protein at 85 days of gestation to 2·34 ± 0·10 nmol/min per mg protein at 142 days of gestation (P<0·005, n=15). The apparent Km did not vary significantly between the 85- and 142-day groups. Vmax, however, was significantly greater in the late-gestation group (2·98 ± 0·02 nmol/min per mg protein) than in the mid-gestation group (2·38 ± 0·13 nmol/min per mg protein, P<0·05). In association with labour, placental LAT activity decreased by 16% (1·96 ± 0·13 nmol/min per mg protein) when compared with that observed in tissue obtained from the non-labouring ewe (P<0·01). The data obtained are consistent with the hypothesis that changes in LAT activity in ovine placenta do not contribute to the gestational increase in prostaglandin formation, but a contribution to the labour-associated increase in non-esterified arachidonic acid availability and eicosanoid formation cannot be negated.

Journal of Endocrinology (1996) 148, 241–247
Transcriptional and post-translational regulation of insulin-like growth factor-binding protein-5 in rat articular chondrocytes
Authors: Matsumoto T , Gargosky S E , Oh Y , Rosenfeld R G
Published: December 2004

The aim of this study was to assess the regulation of insulin-like growth factor-binding proteins (IGFBPs) by IGFs in primary cultures of rat articular chondrocytes (RAC). Employing Western ligand blotting, immunoprecipitation and Northern blot analysis, RAC were found to secrete IGFBP-5 (29 kDa) and IGFBP-4 (24 kDa) as the predominant IGFBPs, as well as IGFBP-2 (32–30 kDa) and IGFBP-3 (43–39 kDa) as the minor species. Treatment of cells with IGF-I and IGF-II resulted in a dose-dependent increase of IGFBP-5 and a small increase in IGFBP-4 in conditioned media (CM). Des(1–3) IGF-I and [Gln6, Ala7,Tyr18, Leu19] IGF-II ([QAYL] IGF-II), which bind to the type 1 IGF receptor but not to IGFBPs, also induced IGFBP-5 peptide, although the increase was less than with IGF-I or IGF-II treatment of RAC. [Leu27] IGF-II, which does not bind to the type 1 IGF receptor but binds to IGFBPs, resulted in little induction of IGFBP-5, while [QAYL-Leu27] IGF-II, which has reduced affinity for both the type 1 IGF receptor and IGFBPs, did not increase IGFBP-5. These data suggest that the increase in IGFBP-5 in CM is modulated by both the type 1 IGF receptor and the interaction between IGFs and IGFBPs. Northern blotting analysis showed that IGF-I, IGF-II and des(1–3) IGF-I treatment of RAC increased steady state levels of IGFBP-5 mRNA, suggesting that the IGF-mediated increase in IGFBP-5 is transcriptionally modulated. Interestingly, the increase in IGFBP-5 peptide levels and mRNA were not parallel, suggesting the possibility of post-translational modifications of IGFBP-5, such as those seen with IGFBP-5 protease. IGFBP-5 protease activity was detectable in untreated CM, whereas treatment with IGF-I and IGF-II partially protected IGFBP-5 from proteolysis. In summary, treatment of RAC with IGF-I and IGF-II results in dose-dependent increases in both IGFBP-5 peptide in the CM and mRNA levels. These changes are mediated by interactions via the type 1 IGF receptor as well as IGFBPs, both transcriptionally and post-translationally.

Journal of Endocrinology (1996) 148, 355–369
Why so much oestriol? A comparison of the aromatisation of androstenedione and 16α-hydroxyandrostenedione when incubated alone or together with human placental microsomes
Authors: Othman Y S H , Oakey R E
Published: December 2004

Estimates of the relative abundance of 16α-hydroxy- and 16-deoxyoestrogens in late pregnancy urine lie between 13:1 and 5:1, yet the ratio of the concentrations of the major precursors 16α-hydroxydehydroepiandrosterone sulphate and dehydroepiandrosterone sulphate in cord blood is about 2·5:1. This discrepancy might imply that 16α-hydroxy-C19 steroids are used more efficiently for placental oestrogen biosynthesis than are the 16α-deoxy-C19 steroids.

On testing this hypothesis by incubation of placental microsomes with 16α-hydroxy- and 16-deoxy- precursors together (concentration ratios 128:1 to 1:1), initial rates of oestrogen formation were highest from the 16-deoxy-C19 steroid. Additionally, whilst each substrate appeared to inhibit the aromatisation of the other, the 16-deoxy-C19 steroid was the more potent inhibitor. These findings were supported by an analogous experiment with placental slices.

When each precursor was examined separately with microsomes from 4 placentae, aromatisation of the 16α-hydroxy-C19 steroid (Michaelis constant, (Km) 0·75–1·24 μmol/l, maximum reaction velocity (Vmax) 28–69 pmol product/min/mg protein) was less efficient than that of the 16-deoxy-C19 steroid (Km 0·10–0·15 μmol/l, Vmax 71–145 pmol product/min/mg protein).

To reconcile the disparity between the measured utilisation of precursors in vitro and expectations drawn from precursor availability and urinary excretion rates, sources of urinary 16α-hydroxyoestrogens additional to placental aromatisation need to be considered. Hydroxylation of 16-deoxyoestrogens (the phenolic pathway) appears limited but aromatisation in fetal liver of 16α-hydroxyandrostenedione not utilised by the placenta appears to be worth attention.

Journal of Endocrinology (1996) 148, 399–407
Melanin-concentrating hormone gene-related peptide stimulates ACTH, but not α-MSH, release from the tilapia pituitary
Authors: Gröneveld Diet , Balm Paul H M , Wendelaar Bonga Sjoerd E
Published: December 2004

Tilapia (Oreochromis mossambicus; teleostei) melanin-concentrating hormone gene-related peptide (tMgrp) was tested for tropic actions on adenocorticotropin hormone (ACTH) and α-melanocyte stimulating hormone (α-MSH) producing cells in the tilapia pituitary gland in vitro. Up to 100 μM synthetic tilapia Mgrp (tMgrp) had no effect on α-MSH release from tilapia neurointermediate lobes in a superfusion set up. However, at concentrations above 1 μM, tMgrp concentration dependently stimulated ACTH release from tilapia anterior lobes. This is the first evidence that Mgrp modulates ACTH release from teleost corticotropes, and this might implicate the peptide in the regulation of the pituitary-interrenal axis of fish.
Pituitary lactotrope expresses transforming growth factor β (TGFβ) type II receptor mRNA and protein and contains 125I-TGFβ1 binding sites
Authors: De A , Morgan T E , Speth R C , Boyadjieva N , Sarkar D K
Published: December 2004

Transforming growth factor β1 (TGFβ1) has recently been shown to be produced in the prolactin (PRL)-secreting lactotropes of the pituitary gland. TGFβ1 inhibits lactotropic secretion and proliferation, and the production of TGFβ1 in lactotropes is reduced during lactotropic growth following estrogen treatment in ovariectomized rats. In many estrogen-responsive tissues, TGFβ1 has been shown to exert its effect by binding to TGFβ1 type II receptors (TβR II) at the cell surface. In this study, we sought to ascertain whether TβR II is involved in TGFβ1 action on lactotropes by determining the changes of TβR II mRNA and protein levels and specific 125I-TGFβ1 binding sites on the lactotropes during estrogen-induced proliferation of lactotropes in Fischer 344 rats. Double immunohistochemical procedures were employed to identify immunoreactive TβR II in PRL-reactive cells. The majority of TβR II-reactive cells in the anterior pituitary were observed to be lactotropes. Dual immunohistochemistry and in situ hybridization procedures also indicated that lactotropes were the major cell types containing TβR II mRNA hybrids. Both the levels of immunoreactive TβR II protein and in situ TβR II mRNA hybrids in the pituitary were significantly decreased in ovariectomized rats after 15 days of estrogen treatment. Determination of 125I-TGFβ1 binding sites in lactotropes by double immunohistochemistry and receptor autoradiography also revealed specific binding sites of 125I-TGFβ1 in lactotropes in the anterior pituitary. 125I-TGFβ1 binding in the anterior pituitary was also reduced following estrogen treatment in ovariectomized rats. These data suggest that down-regulation of TβR II may be an important mechanism of estrogen action on lactotropic cell growth and PRL secretion, and further support the notion that TGFβ1 controls lactotropic function by autocrine/paracrine mechanisms.

Journal of Endocrinology (1996) 149, 19–27
Expression of insulin-like growth factors in two bovine oviductal cultures employed for embryo co-culture
Authors: Xia P , Han V K M , Viuff D , Armstrong D T , Watson A J
Published: December 2004

We have investigated the patterns of expression and cellular localization of polypeptides and mRNAs encoding IGF-I and IGF-II in intact bovine oviduct and two bovine oviductal primary cultures (monolayers and vesicles) which are utilized for supporting development in vitro. IGF-I and IGF-II polypeptides were localized by immunocytochemistry in intact oviduct and in both primary cultures for an 8-day culture interval, but IGF-II polypeptide displayed a more restricted distribution in day 8 monolayer cultures. IGF-I and IGF-II mRNAs were localized in both oviductal cell cultures as assessed by in situ hybridization. We were unable to detect IGF-I and IGF-II mRNAs in intact oviduct by in situ hybridization; however, transcripts encoding IGF-I and IGF-II mRNAs were detected in intact oviduct cell preparations and all primary culture samples by reverse transcription-PCR methods. The origin and phenotypic stability of these cultures was assessed by immunostaining with antibodies raised against vimentin (mesenchymal cell marker) and cytokeratin (epithelial cell marker). Over the culture period, the proportion of vimentin-immunoreactive cells increased in the monolayer cultures but remained at a low level in the vesicle cultures which were predominantly composed of cytokeratin-positive cells. The results suggest that oviductal cell co-culture may facilitate early mammalian development, in part, by the establishment of paracrine growth factor circuits.

Journal of Endocrinology (1996) 149, 41–53
GH regulation of the Type 2 IGF receptor in regenerating skeletal muscle of rats
Authors: Kirk S P , Whittle M A , Oldham J M , Dobbie P M , Bass J J
Published: December 2004

GH enhances skeletal muscle growth, and IGF-II peptide is highly expressed during regeneration. We have therefore investigated the effect of GH administration on IGF-II binding and expression in regenerating rat skeletal muscle using the techniques of receptor autoradiography and in situ hybridisation. Notexin, a myotoxin, was injected into the right M. biceps femoris (day 0), causing affected fibres to undergo necrosis followed by rapid regeneration. Animals were administered either GH (200 μg/100 g body weight) or saline vehicle daily. Contralateral muscles were used as regeneration controls. GH administration during regeneration resulted in significant increases in body weight, and damaged and undamaged muscle weights (P<0·001). IGF-II expression, which was examined in regenerating fibres, survivor fibres and undamaged fibres, varied according to tissue type (P< 0·001). Specifically, IGF-II expression in regenerating fibres was elevated relative to control and survivor fibres after day 3 (P<0·05), with a peak on day 9 (P<0·001). GH did not affect IGF-II message levels. 125I-IGF-II binding in regenerating muscle was examined in the same fibre types as well as in connective tissue. 125I-IGF-II binding in regenerating fibres was higher (P<0·001) than in other tissue types on day 5. GH administration increased 125I-IGF-II binding in all damaged muscle tissues on day 5 (P<0·001, regenerating fibres; P<0·01, others). We believe that this shows for the first time an effect of GH on the Type 2 IGF receptor in regenerating skeletal muscle.

Journal of Endocrinology (1996) 149, 81–91
Parathyroid hormone induces superoxide anion burst in the osteoclast: evidence for the direct instantaneous activation of the osteoclast by the hormone
Authors: Datta H K , Rathod H , Manning P , Turnbull Y , McNeil C J
Published: December 2004

We have shown that superoxide anion (O2−) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2− production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2− generation. The determination of O2− generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2·8) was 104-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2− generation which was unaffected by the presence of ROS 17/2·8 cells. The osteoclasts cocultured with ROS 17/2·8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2− than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2− generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2−; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.

Journal of Endocrinology (1996) 149, 269–275
Gender differences in the associations between cortisol and insulin in healthy subjects
Authors: Stolk R P , Lamberts S W J , de Jong F H , Pols H A P , Grobbee D E
Published: December 2004

To investigate the role of cortisol in the etiology of insulin resistance in men and women, we examined 218 healthy non-hospitalized elderly, selected from the Rotterdam Study. Free cortisol was assessed by the ratio of fasting serum cortisol over corticosteroid-binding globulin (CBG), and insulin resistance was estimated by the fasting insulin level. CBG was higher in women and decreased with age. In both men and women, the early morning free cortisol level showed no association with age or waist/hip ratio. In men, an inverse association between cortisol and body mass index was observed. In women, higher cortisol levels were associated with increased insulin levels; an increase of 9·7 mU/l insulin per unit cortisol/CBG (s.e. 3·9, P=0·01). The association did not change after adjustment for age, body mass index or waist/hip ratio. The results of this study in elderly subjects suggest that in women cortisol may be implicated in the age-associated insulin resistance.

Journal of Endocrinology (1996) 149, 313–318
IGF-I potentiates interleukin-2 production in human peripheral T cells
Authors: Kooijman R , Rijkers G T , Zegers B J M
Published: December 2004

IGF-I stimulates the proliferation and differentiation of many cell types. In the case of T cells, IGF-I has been described to potentiate mitogen-induced DNA synthesis. We have addressed the working mechanism of IGF-I on T cell proliferation by measuring the effects of IGF-I on various stages of T cell activation. We found that IGF-I augmented the phytohaemagglutinin- and anti-CD3-induced interleukin-2 (IL2) production of human peripheral T cells before they enter the S phase of the cell cycle. Furthermore, the addition of IGF-I did not influence DNA synthesis of IL2-dependent growing T cells.

Journal of Endocrinology (1996) 149, 351–356
Differential effects of malnutrition, bile duct ligation and galactosamine injection in young rats on serum levels and gene expression of IGF-binding proteins
Authors: Holt R I G , Baker A J , Jones J S , Crossey P A , Stone N M , Preedy V R , Miell J P
Published: December 2004

Hepatic gene expression and circulating levels of IGF-binding proteins (IGFBP)-1 to -4 were examined in two rat models of liver disease employing acute hepatitis or chronic structural damage.

The study comprised four groups: group 1 (n=6) was injected intraperitoneally with saline and food was available ad libitum (AL), group 2 (n=6) underwent bile duct ligation (BDL), group 3 (n=6) was injected with 400 mg galactosamine (GAL), group 4 (n=6) was sham-operated and pair-fed to group 2 (PF). All were killed by decapitation at day 7.

Serum IGF-I, by RIA, was significantly (P<0·05) lower in the BDL group (458 ± 37 μg/l) and PF group (451 ±51 μg/l) compared with the AL group (643 ± 77 μg/l) and GAL group (720 ± 67 μg/l). Immunoblotting showed raised IGFBP-2 levels in all groups compared with AL (BDL, 167 ± 14% of AL; GAL, 173 ± 13%; PF, 149 ± 9%). IGFBP-3 was decreased in the GAL (56 ± 11%) and PF groups (66 ± 5%) but increased in the BDL group (154 ± 29%). IGFBP-4 was decreased in the GAL (76 ± 11%) and PF groups (47 ± 5%) but unchanged in the BDL group (90 ± 10%).

By Northern analysis, IGFBP-1 mRNA expression was increased in the GAL (321 ± 51%) and PF groups (263 ± 12%) but reduced in the BDL group (68 ± 8%). IGFBP-2 expression increased in all groups (PF, 836 ± 19%; BDL, 683 ± 121%; GAL, 372 ± 68%) and was highest in the BDL and PF groups. IGFBP-3 expression was reduced in all groups (BDL, 57 ± 16%; GAL, 52 ± 12% PF, 51 ± 13%). IGFBP-4 expression was reduced in the GAL (30 ± 4%) and PF (28 ± 5%) groups but unchanged in the BDL group (76 ± 9%).

Marked changes in gene expression of IGFBPs occurred in both models of liver disease, together with serum changes, which were different from each other and from malnutrition alone.

Journal of Endocrinology (1996) 149, 465–472