bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasoactive Intestinal Peptide Is an Important Endocrine Regulatory Factor of Fetal Rat Testicular Steroidogenesis*
Authors: El-Gehani Faraj , Tena-Sempere Manuel , Huhtaniemi Ilpo
Published: January 2014

This study elaborates our recent preliminary finding that vasoactive intestinal peptide (VIP) has a specific stimulatory effect on fetal rat Leydig cells. We examined the dose-response relationship for the effect of VIP on cAMP and testosterone production by dispersed fetal Leydig cells isolated from rat testes on embryonic day (E) 18.5. Further, we used RT-PCR to examine the expression of the VIP gene in fetal brain and testes and that of the VIP receptor genes in fetal testes and used RIA to measure VIP in testes and plasma during the fetal period. VIP stimulated fetal testicular cAMP production at a dose of 10−9 mol/liter, whereas a dose as low as 10−12 mol/liter stimulated testosterone production. This suggests that VIP at low doses may stimulate testosterone production using second messenger pathways other than cAMP. RT-PCR analysis could not reveal either VIP messenger RNA (mRNA) in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, whereas VIP2 receptor mRNA was detected in fetal testes as early as E15.5. Northern hybridization analysis showed that the level of expression of VIP2 receptor mRNA is very low in fetal and neonatal testes and increases with age. The testicular VIP content was unmeasurable by our RIA method (i.e. <1 fmol/testis), whereas the circulating level of VIP was 82.9 ± 1.1 pmol/liter on E17.5 and decreased with advancing fetal age.

In conclusion, our results suggest that VIP from an extratesticular source, possibly from the maternal compartment, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5. These findings may be of physiological significance, because the onset of fetal testicular steroidogenesis occurs at an age (E15.5–19.5) before the onset of pituitary LH secretion.
Clathrin Gene Expression Is Androgen Regulated in the Prostate*
Authors: Prescott James L. , Tindall Donald J.
Published: January 2014

Androgens are required for the development and function of the prostate. In a normal human prostate, androgens control the synthesis of proteins such as prostate-specific antigen and human glandular kallikrein. The prostate secretes these proteins as well as a number of other compounds to form the prostatic fluid. Using differential display PCR to detect novel androgen-regulated genes, clathrin heavy chain expression was identified as potentially being up-regulated by androgens in the prostate cancer cell line LNCaP. We report here that the clathrin heavy chain and light chain genes are regulated by androgens. Clathrin heavy chain messenger RNA was up-regulated by androgens in a concentration- and time-specific manner in the LNCaP cell line. Translation of clathrin heavy chain messenger RNA was stimulated by androgens. Steady state levels of clathrin light chains a and b were up-regulated in the presence of androgen in LNCaP cells. Clathrin gene expression was examined in normal rat prostates, and similar results were found. Clathrin heavy chain protein levels in the rat prostate are also affected by the androgen status of the animal. We hypothesize that clathrin may be involved in the exocytosis of androgen-regulated secretory proteins such as prostate-specific antigen and human glandular kallikrein.
Expression of Retinoic Acid Receptor-β Sensitizes Prostate Cancer Cells to Growth Inhibition Mediated by Combinations of Retinoids and a 19-nor Hexafluoride Vitamin D3 Analog*
Authors: Campbell Moray J. , Park Susan , Uskokovic Milan R. , Dawson Marcia I. , Koeffler H. Phillip
Published: January 2014

Retinoids and analogs of vitamin D3 may achieve greater in vivo applications if the toxic side effects encountered at pharmacologically active doses could be alleviated. These seco-steroid hormones often act in concert, and therefore, we attempted to dissect these interactions by isolating combinations of receptor-selective retinoids and a potent vitamin D3 analog[ 1α,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3, code name LH] that were potent inhibitors of prostate cancer cell growth at low, physiologically safer doses.

Using a panel of prostate cancer cell lines representing progressively more transformed phenotypes, we found that the LNCaP cell line (least transformed) was either additively or synergistically inhibited in its clonal growth by LH and various naturally occurring and receptor-selective retinoids, the most potent combination being with a retinoic acid receptor (RAR)βγ-selective retinoid (SR11262). The effect was not found with either PC-3 (intermediate transformation) or DU-145 (most transformed). We also undertook RT-PCR to examine the subtypes of RARs present, and we found that PC-3 and DU-145 did not express RARβ. Stable expression of RARβ into the RARβ-negative PC-3 cells resulted in increased sensitivity to SR11262 and LH proportional to the amount of RARβ expressed.

This study indicates that RARβ may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.
Effects of Sex Steroids on Secretory Granule Formation in Gonadotropes of Castrated Male Rats with Respect to Granin Expression*
Authors: Watanabe Tsuyoshi , Banno Tomohiro , Jeziorowski Thomas , Ohsawa Yoshiyuki , Waguri Satoshi , Grube Dietrich , Uchiyama Yasuo
Published: January 2014

Pituitary gonadotropes show sex-related differences in their ultrastructure. Typical gonadotropes of male rats exhibit both large granules, which contain chromogranin A (CgA), and small granules, which contain secretogranin II (SgII). In contrast, typical female rat gonadotropes show only a very few large granules among the numerous small granules. To clarify the nature of the biogenesis of these secretory granules and the effects of sex steroids, the ultrastructural and immunocytochemical changes in gonadotropes were examined in castrated male rats supplied with a testosterone or estradiol implant. In castrated rats, pituitary expression and plasma levels of LH increased drastically, but the pituitary content of CgA decreased. The majority of gonadotropes then showed features of “castration cells” containing many small secretory granules. A testosterone implant to castrated rats remarkably suppressed the expression and circulating levels of LH and increased the CgA content in the pituitary to near-normal levels. In this situation, immunocytochemical studies demonstrated that gonadotropes again exhibited large and small secretory granules with the respective localization of CgA and SgII. On the contrary, in castrated rats supplied with an estradiol implant, the expression and content of CgA in the pituitary were remarkably suppressed, and large secretory granules disappeared from gonadotropes. These results suggest that the expression of CgA in gonadotropes is regulated differently by male and female sex steroids. These different effects of androgen and estrogen on the expression level of CgA are closely associated with the sex-related differences in the ultrastructure of secretory granules within gonadotropes.
Thyrotropin-Releasing Hormone Gene Expression in the Anterior Pituitary. IV. Evidence for Paracrine and Autocrine Regulation
Authors: Bruhn Thomas O. , Rondeel Jan M. M. , Jackson Ivor M. D.
Published: January 2014

Disulfiram (Dis), an inhibitor of peptidyl-glycine α-amidating monooxygenase, the enzyme responsible for the production ofα -amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated.

Dis (8 μm) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nm), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (−73%) and TSH (−40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion.

Dis significantly elevated prepro-TRH (25–50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.
Epidermal Growth Factor-Induced Heterologous Desensitization of the Luteinizing Hormone/Choriogonadotopin Receptor in a Cell-Free Membrane Preparation Is Associated with the Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor**This work was supported by USDA Grant NRICGP-9401432 (to M.H.D.).
Authors: Lamm Marilyn L. G. , Rajagopalan-Gupta Rajsree M. , Hunzicker-Dunn Mary
Published: January 2014

Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Giα, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.
Stimulation of Osteoprotegerin Ligand and Inhibition of Osteoprotegerin Production by Glucocorticoids in Human Osteoblastic Lineage Cells: Potential Paracrine Mechanisms of Glucocorticoid-Induced Osteoporosis1
Authors: Hofbauer Lorenz C. , Gori Francesca , Riggs B. Lawrence , Lacey David L. , Dunstan Colin R. , Spelsberg Thomas C. , Khosla Sundeep
Published: January 2014

Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG-L in various human osteoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70–90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose dependently (10−10m–10−7m) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 ± 0.02 ng/ml (control) to 0.30 ± 0.01 ng/ml (88% inhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectively. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony-stimulating factor). Moreover, dexamethasone (10−8m) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocorticoids promote osteoclastogenesis by inhibiting OPG and concurrently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.
Breast Cancer Cells Interact with Osteoblasts to Support Osteoclast Formation1
Authors: Thomas Rachel J. , Guise Theresa A. , Yin Juan Juan , Elliott Jan , Horwood Nicole J. , Martin T. John , Gillespie Matthew T.
Published: January 2014

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption. Recently the osteoclast differentiation factor (ODF), better termed RANKL (receptor activator of NF-κB ligand), expressed by osteoblasts has been cloned as well as its cognate signaling receptor, receptor activator of NFκB (RANK), and a secreted decoy receptor osteoprotegerin (OPG) that limits RANKL’s biological action. We determined that the breast cancer cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast cancers do not express RANKL but express OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo required exogenous agents for osteoclast formation. When MCF-7 cells overexpressing PTHrP were cultured with murine osteoblasts, osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no effect on RANKL or OPG mRNA levels when cultured with osteoblastic cells. Using a murine model of breast cancer metastasis to bone, we established that MCF-7 cells that overexpress PTHrP caused significantly more bone metastases, which were associated with increased osteoclast formation, elevated plasma PTHrP concentrations and hypercalcaemia compared with parental or empty vector controls.
Three Novel Mutations at Serine 314 in the Thyroid Hormone β Receptor Differentially Impair Ligand Binding in the Syndrome of Resistance to Thyroid Hormone1
Authors: Gurnell M. , Rajanayagam O. , Agostini M. , Clifton-Bligh R. J. D. , Wang T. , Zelissen P. M. J. , van der Horst F. , Wiel A. van de , Macchia E. , Pinchera A. , Schwabe J. W. R. , Chatterjee V. K. K.
Published: January 2014

The syndrome of resistance to thyroid hormone is associated with diverse mutations in the ligand-binding domain of the thyroid hormone β receptor, localizing to three clusters around the hormone binding cavity. Here, we report three novel resistance to thyroid hormone mutations (S314C, S314F, and S314Y), due to different nucleotide substitutions in the same codon, occurring in six separate families. Functional characterization of these mutant receptors showed marked differences in their properties. S314F and S314Y receptor mutants exhibited significant transcriptional impairment in keeping with negligible ligand binding and were potent dominant negative inhibitors of wild-type receptor action. In contrast, the S314C mutant bound ligand with reduced affinity, such that its functional impairment and dominant negative activity manifest at low concentrations of thyroid hormone, but are more reversible at higher T3 concentrations. The degree of functional impairment of mutant receptors in vitro may correlate with the magnitude of thyroid dysfunction in vivo. Modelling these mutations using the crystal structure of thyroid hormone receptor β shows why ligand binding is perturbed and why the phenylalanine/tyrosine mutations are more deleterious than cysteine.
Early Effects of Castration on the Vascular System of the Rat Ventral Prostate Gland*
Authors: Shabisgh Ahmad , Tanji Nozomu , D’Agati Vivette , Burchardt Martin , Rubin Mark , Goluboff Erik T. , Heitjan Daniel , Kiss Alex , Buttyan Ralph
Published: January 2014

Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.
Growth Hormone-Mediated Regulation of Insulin-Like Growth Factor I Promoter Activity in C6 Glioma Cells*
Authors: Benbassat Carlos , Shoba Lungile N. N. , Newman Marsha , Adamo Martin L. , Frank Stuart J. , Lowe William L.
Published: January 2014

The molecular mechanisms by which GH regulates insulin-like growth factor (IGF-I) gene expression remain obscure. One difficulty has been the lack of established GH-responsive cell lines that express the IGF-I gene. To develop such a cell line, we used rat C6 glioma cells which, as determined by RNase protection assay, express the IGF-I gene but not the GH receptor gene. To confer GH responsiveness, C6 cells were cotransfected with vectors that express the GH receptor (pRc/CMV WTrGHR) and Jak2 (pRc/CMV Jak2). GH responsiveness was demonstrated using luciferase reporter genes containing either the Sis-inducible element from the c-fos gene (pTK81-SIE-Luc) or 6 copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc). The SIE is activated by binding of STAT1 and 3, whereas the GLE binds STAT5. In cells cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and either pTK81-SIE-Luc or pSpi GLE-Luc, treatment with 500 ng/ml GH for 24 h stimulated a 3.1- and 1.7-fold increase in luciferase activity, respectively. These data suggest that in C6 cells cotransfected with pRc/CMV WTrGHR and pRc/CMV Jak2, GH activates STAT1, 3, and 5. To determine whether GH-responsive IGF-I promoter activity could be demonstrated, C6 cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2, and an IGF-I-luciferase fusion gene that contained a fragment of the rat IGF-I gene that extended from −412 in the 5′-flanking region of exon 1 to the Met-22 in exon 3. GH stimulated a modest, but reproducible, 1.7-fold increase in luciferase activity in these cells, suggesting that a GH-responsive element is present in this region of the IGF-I gene. To better localize the GH-responsive element, cells were cotransfected with pRc/CMV WTrGHR, pRc/CMV Jak2 plus one of several IGF-I-luciferase fusion genes containing either fragments of one of the two promoters in the IGF-I gene or a fragment of intron 2 that includes a GH-responsive DNase I hypersensitivity site. For all constructs, treatment with GH for 24 h did not stimulate a significant increase in luciferase activity, suggesting that GH-responsive sequences are not located in these specific regions of the IGF-I gene or that GH-directed transcription of the IGF-I gene is mediated via several different regions of the IGF-I gene and the effect of any one of these regions in isolation was not sufficiently robust to be detected in this model system. In summary, transient expression of the GH receptor and Jak2 in C6 cells creates a GH-responsive system that activates STAT1, 3, and 5. Moreover, a fragment of the IGF-I gene that contains exons 1 and 2, a fragment of exon 3, and introns 1 and 2 is GH responsive using this model system.
Evidence That the Inhibition of Luteinizing Hormone Secretion Exerted by Central Administration of Neuropeptide Y (NPY) in the Rat Is Predominantly Mediated by the NPY-Y5 Receptor Subtype1
Authors: Raposinho Paula D. , Broqua Pierre , Pierroz Dominique D. , Hayward Amanda , Dumont Yvan , Quirion Remi , Junien Jean-Louis , Aubert Michel L.
Published: January 2014

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7–2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist[ Leu31,Pro34]NPY (0.7–2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7–7 nmol) as well as the mixed Y2-Y5 agonist PYY3–36 (0.7–7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion.[ d-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6–100 μg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 μg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3–36, human PP,[ d-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific[ 125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of[ d-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.
Role of Gonadotropin-Releasing Hormone as an Autocrine Growth Factor in Human Ovarian Surface Epithelium1
Authors: Kang Sung Keun , Choi Kyung-Chul , Cheng Kwai Wa , Nathwani Parimal S. , Auersperg Nelly , Leung Peter C. K.
Published: January 2014

Epithelial ovarian cancer, which accounts for 80–90% of all ovarian cancers, is the most common cause of death from gynecological malignancies and is believed to originate from the ovarian surface epithelium. In the present study we investigated the expression of GnRH and its receptor in human ovarian surface epithelial (hOSE) cells and provided novel evidence that GnRH may have antiproliferative effects in this tissue. Using RT-PCR and Southern blot analysis, we cloned the GnRH and GnRH receptor (GnRHR) in hOSE cells. Sequence analysis revealed that GnRH and its receptor have sequences identical to those found in the hypothalamus and pituitary, respectively. To address whether GnRH regulates its own and receptor messenger RNA (mRNA), the cells were treated with different concentrations of the GnRH agonist (d-Ala6)-GnRH. Expression levels of GnRH and its receptor were investigated using quantitative and competitive RT-PCR, respectively. Interestingly, a biphasic effect was observed for the GnRH and GnRHR mRNA levels. High concentrations of the GnRH agonist (10−7 and 10−9m) decreased GnRH and GnRHR mRNA levels, whereas a low concentration (10−11m) resulted in up-regulation of GnRH and receptor mRNA levels. Treatment with the GnRH antagonist, antide, prevented the biphasic effects of the GnRH agonist in hOSE cells, confirming the specificity of the response. Furthermore, to investigate the physiological significance, we studied receptor-mediated growth regulatory effects of GnRH in human ovarian surface epithelial cells. The cells were treated with GnRH analogs, and the proliferative index of cells was measured using a [3H]thymidine incorporation assay. (d-Ala6)-GnRH had a direct inhibitory effect on the growth of hOSE cells in a time- and dose-dependent manner. This antiproliferative effect of the GnRH agonist was receptor mediated, as cotreatment of hOSE cells with antide abolished the growth inhibitory effects of the GnRH agonist. The results strongly suggest that GnRH can act as an autocrine/paracrine regulator in hOSE cells.
Calcitriol-Induced Apoptosis in LNCaP Cells Is Blocked By
Overexpression of Bcl-21
Authors: Blutt Sarah E. , McDonnell Timothy J. , Polek Tara C. , Weigel Nancy L.
Published: January 2014

While the role of vitamin D in bone and mineral metabolism has been investigated extensively, the role of the vitamin D receptor in other tissues is less well understood. 1,25-dihydroxyvitamin D3 (calcitriol) can act as a differentiating agent in normal tissues and can inhibit the growth of many cancer cell lines including LNCaP prostate cancer cells. We have shown previously that calcitriol causes LNCaP cell accumulation in the G0/G1 phase of the cell cycle. In this study, we demonstrate that calcitriol also induces apoptosis of LNCaP cells. The calcitriol-induced apoptosis is accompanied by a down-regulation of Bcl-2 and Bcl-XL proteins, both of which protect cells from undergoing apoptosis. Other proteins important in apoptotic control, Bax, Mcl-1, and Bcl-Xs, are unaffected by calcitriol treatment. We find that overexpression of Bcl-2 blocks calcitriol-induced apoptosis and reduces, but does not eliminate, calcitriol-induced growth inhibition. We conclude that both regulation of cell cycle and the apoptotic pathway are involved in calcitriol action in prostate cancer cells.
Resveratrol Acts as a Mixed Agonist/Antagonist for Estrogen Receptors α and β*
Authors: Bowers Jennifer L. , Tyulmenkov Valentyn V. , Jernigan Sarah C. , Klinge Carolyn M.
Published: January 2014

Epidemiological evidence indicates that phytoestrogens inhibit cancer formation and growth, reduce cholesterol levels, and show benefits in treating osteoporosis. At least some of these activities are mediated through the interaction of phytoestrogens with estrogen receptors α and β (ERα and ERβ). Resveratrol, trans-3,5,4′-trihydroxystilbene, is a phytoestrogen in grapes that is present in red wine. Resveratrol was shown to bind ER in cytosolic extracts from MCF-7 and rat uteri. However, the contribution of ERα vs. ERβ in this binding is unknown. Here we report that resveratrol binds ERβ and ERα with comparable affinity, but with 7,000-fold lower affinity than estradiol (E2). Thus, resveratrol differs from other phytoestrogens that bind ERβ with higher affinity than ERα. Resveratrol acts as an estrogen agonist and stimulates ERE-driven reporter gene activity in CHO-K1 cells expressing either ERα or ERβ. The estrogen agonist activity of resveratrol depends on the ERE sequence and the type of ER. Resveratrol-liganded ERβ has higher transcriptional activity than E2-liganded ERβ at a single palindromic ERE. This indicates that those tissues that uniquely express ERβ or that express higher levels of ERβ than ERα may be more sensitive to resveratrol’s estrogen agonist activity. For the natural, imperfect EREs from the human c-fos, pS2, and progesterone receptor (PR) genes, resveratrol shows activity comparable to that induced by E2. We report that resveratrol exhibits E2 antagonist activity for ERα with select EREs. In contrast, resveratrol shows no E2 antagonist activity with ERβ. These data indicate that resveratrol differentially affects the transcriptional activity of ERα and ERβ in an ERE sequence-dependent manner.
Glucagon-Like Peptide-1 Induces Cell Proliferation and Pancreatic-Duodenum Homeobox-1 Expression and Increases Endocrine Cell Mass in the Pancreas of Old, Glucose-Intolerant Rats
Authors: Perfetti Riccardo , Zhou Jie , Doyle Máire E. , Egan Josephine M.
Published: January 2014

Glucose homeostasis in mammals is maintained by insulin secretion from the β-cells of the islets of Langerhans. Type 2 diabetes results either from primary β-cell failure alone and/or a failure to secrete enough insulin to overcome insulin resistance. Here, we show that continuous infusion of glucagon-like peptide-1 (7–36) (GLP-1; an insulinotropic agent), to young and old animals, had effects on the β-cell of the pancreas other than simply on the insulin secretory apparatus. Our previous studies on a rodent model of glucose intolerance, the aging Wistar rat, show that a plateau in islet size, insulin content, and β-cell mass is reached at 13 months, despite a continuing increase in body weight. Continuous sc infusion of GLP-1 (1.5 pm/kg·min), over 5 days, resulted in normal glucose tolerance. Our current results in both young and old rats demonstrate that treatment caused an up-regulation of pancreatic-duodenum homeobox-1 (PDX-1) expression in islets and total pancreas, induced pancreatic cell proliferation, and β-cell neogenesis. The effects on levels of PDX-1 messenger RNA were abrogated by simultaneous infusion of Exendin (9–39), a specific antagonist of GLP-1. PDX-1 protein levels increased 4-fold in whole pancreata and 6-fold in islets in response to treatment.β -cell mass increased to 7.2 ± 0.58 from 4.88 ± 0.38 mg, treated vs. control, respectively, P< 0.02. Total pancreatic insulin content also increased from 0.55± 0.02 to 1.32 ± 0.11 μg/mg total pancreatic protein. Therefore, GLP-1 would seem to be a unique therapy that can stimulate pancreatic cell proliferation and β-cell differentiation in the pancreas of rodents.
Female Fertility Is Reduced in Mice Lacking Ca2+/Calmodulin-Dependent Protein Kinase IV**This work was supported by an NIH Medical Scientist Training Program award (to J.Y.W.) and NIH Grants HD-07503 (to A.R.M.) and HD-1272 (to J.S.R.)
Authors: Wu Joy Y. , Gonzalez-Robayna Ignacio J. , Richards JoAnne S. , Means Anthony R.
Published: January 2014

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.
Vitamin D Is an Important Factor in Estrogen Biosynthesis of Both Female and Male Gonads*
Authors: Kinuta Keiko , Tanaka Hiroyuki , Moriwake Tadashi , Aya Kunihiko , Kato Shigeaki , Seino Yoshiki
Published: January 2014

In the present study, the role of vitamin D in the regulation of estrogen synthesis in gonads was investigated. Vitamin D receptor null mutant mice showed gonadal insufficiencies. Uterine hypoplasia and impaired folliculogenesis were observed in the female, and decreased sperm count and decreased motility with histological abnormality of the testis were observed in the male. The aromatase activities in these mice were low in the ovary, testis, and epididymis at 24%, 58%, and 35% of the wild-type values, respectively. The gene expression of aromatase was also reduced in these organs. Elevated serum levels of LH and FSH revealed hypergonadotropic hypogonadism in these mice. The gene expressions of estrogen receptor α and β were normal in gonads in these mice. Supplementation of estradiol normalized histological abnormality in the male gonads as well as in the female. Calcium supplementation increased aromatase activity and partially corrected the hypogonadism. When the serum calcium concentration was kept in the normal range by supplementation, the aromatase activity in the ovary increased to 60% of the wild-type level, but LH and FSH levels were still elevated. These results indicated that vitamin D is essential for full gonadal function in both sexes. The action of vitamin D on estrogen biosynthesis was partially explained by maintaining calcium homeostasis; however, direct regulation of the expression of the aromatase gene should not be neglected.
Knockout of the Murine Prostaglandin EP2 Receptor Impairs Osteoclastogenesis in Vitro*
Authors: Li Xiaodong , Okada Yosuke , Pilbeam Carol C. , Lorenzo Joseph A. , Kennedy Christopher R. J. , Breyer Richard M. , Raisz Lawrence G.
Published: January 2014

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (±) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 −/−) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 −/− animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 −/− cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2− /− mice, by 88% or 68% when only osteoblastic cells were from EP2 −/− mice and by 58% or 35% when only spleen cells were from EP2 −/− mice. PGE2 increased receptor activator of nuclear factor (NF)-kB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 −/− mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 −/− mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2+ /+ mice but had little effect on cells from EP2 −/− mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 −/− mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 −/− mice.
Localization of 17β-Hydroxysteroid Dehydrogenase/17-Ketosteroid Reductase Isoform Expression in the Developing Mouse Testis—Androstenedione Is the Major Androgen Secreted by Fetal/Neonatal Leydig Cells*
Authors: O’Shaughnessy P. J. , Baker P. J. , Heikkilä M. , Vainio S. , McMahon A. P.
Published: January 2014

The final step in the biosynthesis of testosterone is reduction of androstenedione by the enzyme 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase (17βHSD/17KSR). In this study, we have examined expression of the four known reductive isoforms of 17βHSD/17KSR (types 1, 3, 5, and 7) in the developing mouse testis and have determined changes in the localization of isoform expression and testosterone secretion during development. Using RT-PCR isoforms 1, 3, and 7 were shown to be expressed in the seminiferous tubules of neonatal testis, whereas isoforms 3 and 7 were expressed in the interstitial tissue of the adult testis. The type 7 isoform is unlikely to be involved in androgen synthesis and further study concentrated on the type 3 isoform. Developmentally, isoform type 3 was expressed in the seminiferous tubules up to day 10, showed little or no expression on day 20 and from day 30 was confined to the interstitial tissue. In situ hybridization confirmed that the type 3 isoform was expressed only in the seminiferous tubules in fetal testes and in the interstitial tissue in adult testes. In accordance with the localization of enzyme messenger RNA expression 17-ketosteroid reductase enzyme activity was very low in isolated interstitial tissue from neonatal testes while interstitial tissue from adult testes showed high activity. Seminiferous tubules from both neonatal and adult testes showed high levels of enzyme activity. The major androgen secreted by the interstitial tissue of prepubertal animals was androstenedione up to day 20 while 5α-androstanediol and/or testosterone were the major androgens secreted from day 30 onwards. These results show that fetal Leydig cells do not express significant levels of a reductive isoform of 17βHSD/17KSR and that androstenedione is the major androgen secreted by these cells. Production of testosterone up until puberty is dependent upon 17βHSD/17KSR activity in the seminiferous tubules—a“ two cell” requirement for testosterone synthesis. Expression of the 17βHSD/17KSR type 3 isoform (the main reductive isoform in the testis) declines in the seminiferous tubules before puberty but then reappears in the developing adult Leydig cell population.
4-Hydroxytamoxifen Trans-Represses Nuclear Factor-κB Activity in Human Osteoblastic U2-OS Cells through Estrogen Receptor (ER)α, and Not through ERβ*
Authors: Quaedackers Monique E. , Van Den Brink Christina E. , Wissink Sacha , Schreurs Richard H. M. M. , Gustafsson Jan-Åke , Van Der Saag Paul T. , Van Der Burg Bart
Published: January 2014

Estrogens are important mediators of bone homeostasis, and postmenopausal estrogen replacement therapy is extensively used to prevent osteoporosis. The biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)α and ERβ. ERα, not only trans-activates target genes in a hormone-specific fashion, but it can also neutralize other transcriptional activators, such as nuclear factor (NF)-κB, causing repression of their target genes. A major mechanism by which estrogens prevent osteoporosis seems to be repression of transcription of NF-κB target genes, such as the osteoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ERs in repression of NF-κB signaling in bone cells, we first carried out transient transfections with ERα or ERβ of the human osteoblastic U2-OS cell line, in which endogenous NF-κB was stimulated by tumor necrosis factor α. Repression by ERα was already observed without 17β-estradiol, whereas addition of the ligand increased repression to 90%. ERβ, however, was able to repress NF-κB activity only in the presence of ligand. Because it is known that some antiestrogens can also display tissue-specific agonistic properties, 4-hydroxytamoxifen was tested for its capacity in repressing NF-κB activity and was found to be active (albeit less efficient than 17β-estradiol) and, interestingly, only with ERα. The pure antagonist ICI 164,384 was incapable of repressing through any ER subtypes. Deletion analysis and the use of receptor ERα/ERβ-chimeras showed that the A/B domain, containing activation function-1, is essential for this suppressive action. Next, we developed stable transfectants of the human osteoblastic U2-OS cell line containing ERα or ERβ in combination with an NF-κB luciferase reporter construct. In these cell lines, repression of NF-κB activity was only mediated through ERα and not through ERβ. These findings offer new insights into the specific role of both ER subtypes in bone homeostasis and could eventually help in developing more specific medical intervention strategies for osteoporosis.
Insulinotropic Hormone Glucagon-Like Peptide 1 (GLP-1) Activation of Insulin Gene Promoter Inhibited by p38 Mitogen-Activated Protein Kinase*
Authors: Kemp Daniel M. , Habener Joel F.
Published: January 2014

The insulin gene promoter contains many transcriptional response elements that predispose the gene to a wide range of regulatory signals. Glucagon-like peptide 1 (GLP-1) stimulates insulin gene transcription by intracellular second messenger cascades leading to direct transcription factor activation or to the up-regulation of insulin promoter specific transcription factors. In these studies, we have identified a novel regulatory signaling mechanism acting on the rat insulin 1 promoter (rINS1) in the INS-1 β-cell line. In the presence of stimulatory concentrations of GLP-1 (0.1–100 nm) on rINS1 activity, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) using SB 203580 resulted in a marked increase in promoter activity (maximum 3-fold) over GLP-1 alone, as determined by rINS1 promoter-luciferase reporter gene expression. This effect was revealed to be mediated via the cAMP response element (CRE) of rINS1, because site directed mutagenesis of the CRE motif in rINS1 abolished the increased response to SB 203580. Furthermore, inhibition of p38 MAPK uncovered a similar, more pronounced, response in the expression of a generic CRE promoter driven reporter gene. Time course dose-response studies indicate that the p38 MAPK induced inhibitory response may involve expression of immediate early genes (IEGs); maximum repression of rINS1 activity occurred after 4 h of treatment, comparable with regulatory responses by IEGs. In conclusion, these results demonstrate a novel signaling mechanism whereby p38 MAPK represses rINS1 promoter activity in response to GLP-1, suggesting the involvement of a robust regulatory control by p38 MAPK in insulin gene expression. The relevance of this mechanism may be most apparent during periods of cellular stress in which p38 MAPK activity is stimulated. In this regard, reduced insulin expression levels caused by chronic hyperglycemia (glucotoxicity) and/or hyperlipidemia (lipotoxicity) may be a direct consequence of this mechanism.
Determination of Three Isoforms of the Receptor Activator of Nuclear Factor-κΒ Ligand and Their Differential Expression in Bone and Thymus*
Authors: Ikeda Tohru , Kasai Michiyuki , Utsuyama Masanori , Hirokawa Katsuiku
Published: January 2014

The receptor activator of nuclear factor (NF)-κB ligand [RANKL; also known as tumor necrosis factor-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-κB (RANK) and act not only as a key factor for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL 2 has a shorter intracellular domain than the original RANKL (RANKL 1), and RANKL 3 lacks a transmembrane domain and was thought to act as a soluble form. In the bone marrow stromal cell line ST2 and preosteoblastic cell line MC3T3-E1, all three RANKL isoforms were detected, but the expression of RANKL 2 was preferentially suppressed by treatment with 1α,25-dihydroxyvitamin D3 and dexamethasone. In young adult thymus, CD4−CD8− double-negative cells were positive for all three isoforms, CD4+CD8+ double-positive cells were positive for RANKL 1 and RANKL 3 but negative for RANKL 2, and CD4+CD8− and CD4−CD8+ single-positive cells were positive for all three isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with each RANKL isoform indicated that the three RANKL isoforms were translated, and RANKL 2 protein predominantly stayed in the endoplasmic reticulum and Golgi networks. These results indicate that there are three kinds of RANKL-RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentiation than previously thought.
Mutagenesis of Basic Amino Acids in the Carboxyl-Terminal Region of Insulin-Like Growth Factor Binding Protein-5 Affects Acid-Labile Subunit Binding
Authors: Firth Sue M. , Clemmons David R. , Baxter Robert C.
Published: January 2014

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 was recently shown to form ternary complexes with insulin-like growth factor (IGF) and the acid-labile subunit (ALS). Previous studies using IGFBP-5/IGFBP-6 chimeric proteins have identified major and minor ALS binding sites in the carboxyl-terminal and central regions, respectively of IGFBP-5. We now report that ALS binds to IGFBP-3 (Ka = 1.1 ± 0.1 liters/nmol) and IGFBP-5 (Ka = 1.8 ± 0.5 liters/nmol) with similar binding affinities. Using site-specific mutants, we have identified residues K211/R214/K217/R218 within the carboxyl-terminal region of IGFBP-5 as being essential for ALS binding. Mutation of K134R136 or K138K139 in the central region of IGFBP-5 resulted in a small decrease in ALS binding.