Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts

In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.     

Rabbit endometrium in organ culture: Morphological evidence for progestational differentiation in vitro

Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.Abbreviations d.p.c. dies post coitum - d p. hCG days after injection of hCG (dies post injectionem hCG) - FBS fetal bovine serum - hCG human chorionic gonadotropin Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984)     

Non-genomic effects of the androgen receptor and vitamin D agonist are involved in suppressing invasive phenotype of prostate cancer cells

Suppression of invasive phenotype is essential in developing new therapeutic tools to treat prostate cancer (PC). Evidence indicates that androgen-dependent (AD) prostate cancer cells are characterized by a lower malignant phenotype. We have demonstrated that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion of these cells through modulation of alpha6beta4 integrin expression, indicating a genotropic effect of androgens in inhibiting invasion ability of AD PC cells. Later on, we have shown that also a non-genotropic mechanism is involved in such an effect. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3-AR cells co-localizes with the EGFR receptors (EGFR) in PC3-AR cells. Co-immunoprecipitation studies both in PC3-AR cells and in the AD cell line LNCaP that physiologically express both receptors, confirm the occurrence of an interaction between of the two proteins. In PC3-AR cells, we demonstrated a disruption of EGFR signalling properties (reduced EGF-induced EGFR autotransphosphorylation, reduced EGF-stimulated PI3K activity as well as EGFR-PI3K interaction) contributing to the lower invasive phenotype of these cells. In another study, we investigated the effects of a new Vitamin D analogue, BXL628, on invasion in response to KGF in the androgen-independent PC cell line DU145. We found that the compound was able to reduce proliferation and invasion of the cells in response to the growth factor. In addition, we found that KGF-induced autotransphosphorylation of KGF receptor (KGFR) and PI3K activation were suppressed after short-term (5min) pre-treatment with the analogue before addition of KGF. Collectively, these studies demonstrate that a non-genotropic effect due to a direct interaction of the androgen receptor with EGFR and to a rapid effect of a Vitamin D agonist on KGFR may disrupt signalling of GF leading to decreased tumorigenicity and a less malignant phenotype of PC cells in vitro.     

(Arene)Cl₂Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: interaction with sex hormone binding globulin and anticancer activity

(Arene)dichloridoruthenium(II) complexes with N-coordinated isonicotinates of androgens (6) and estrogens (9) were prepared and tested for affinity to the estrogen receptor (ERα) and sex hormone binding globulin (SHBG), as well as for cytotoxicity in cancer cells. None of the new complexes bound noticeably to the ER and most of them also bound less strongly to SHBG than the corresponding unmetallated steroids 7. In MTT assays the Ru(p-cymene) complexes 9 of 2-substituted estrones were equally or even more cytotoxic than the metal-free steroids against hormone-dependent (MCF-7 breast and KB-V1 cervix carcinomas) and hormone-independent (518A2 melanoma) cells. The addition of external SHBG to MTT assays lowered the cytotoxicities of the complexes 9 and distinctly more so those of some steroids 7, probably by the way of sequestration and reduction of the cellular uptake. In the absence of SHBG the estrogen complexes 9 were internalized by 518A2 melanoma cells and ruthenated their DNA as quantified by ICP-OES. They also ruthenated salmon sperm DNA but did not change the topology of plasmid DNA in EMSA experiments. In addition, the Ru(p-cymene) complex of 2-ethoxyestrone (9c) was shown to reduce the motility of 518A2 melanoma cells in a wound-healing assay.     

Diazepam binding inhibitor (DBI) reduces testosterone and estradiol levels in vivo

Diazepam binding inhibitor (DBI) is a putative endogenous ligand capable of binding to the central type benzodiazepine (BZD) receptor located on the GABAA receptor and the peripheral type BZD receptor on the mitochondrial outer membrane. We examined the effects of an intracerebroventricular injection of DBI on the serum levels of the gonadal hormones, testosterone and estradiol, respectively, in male and female mice. DBI (0.3-10 nmol/mouse, i.c.v.) significantly reduced the levels of both gonadal hormones in a dose-dependent manner. The decrease in the gonadal hormone levels became evident at 1 hr and lasted for at least 4 hrs after the DBI injection. The effects of DBI (3 nmol/mouse, i.c.v.) in male and female mice were completely attenuated by the coadministration of flumazenil (66 nmol/mouse), a selective antagonist for the central type BZD receptor. These results suggest that DBI acts as an endogenous modulator to regulate the levels of gonadal hormones in vivo, and that the DBI-induced decrease in gonadal hormone levels is mediated by down regulation of the GABAergic system, implicated in gonadotropin-releasing systems and/or the hypothalamic-pituitary-gonadal axis.     

Differential effects of testosterone and progesterone on the activation and retention of courtship behavior in sexual and parthenogenetic whiptail lizards

Both testosterone (T) and progesterone (P) facilitate the expression of male-typical sexual behavior in a variety of animals, including rodents and lizards. In two species of whiptail lizards, Cnemidophorus inornatus and C. uniparens, both hormones elicit the full repertoire of courtship behavior. However, the relative efficacy of the two hormones is unknown. In Experiments 1 and 2 we assessed differences in capacity of exogenous T and P to induce male-typical courtship behavior in gonadectomized whiptail lizards. In both species, individuals implanted with T showed more frequent courtship behavior relative to those implanted with P or cholesterol. In Experiments 3 and 4 we examined whether T and P differentially affected the retention of courtship behavior following implant removal. In both species, individuals implanted with T showed more courtship behavior following implant removal than those previously given P. In these experiments, implants were removed at a time when individuals in both groups were behaviorally similar; therefore, the differences in behavior following implant removal were not due to differences in the amount of courtship experience. Taken together, the hormone that was more effective at activating courtship behavior was also more effective at maintaining courtship behavior following implant removal. In summary, though both T and P can elicit identical sexual behaviors in both whiptail species, T has a greater and more lasting effect on courtship behavior and possibly on the neural circuits underlying courtship behavior.     

雄性大熊猫尿中睾酮和皮质醇代谢物水平及其与繁殖、笼养和季节的关系

收集了雄性大熊猫尿样肾上腺皮质激素和雄性激素代谢物年周期变化基础数据。二月份雄性激素水平最高,与繁殖季节的开始相关。繁殖成功与雄性激素水平无相关,与不同的笼养环境也无相关。与其他熊类物种相比,大熊猫交配和雄性激素水平峰值在一年中的较早时候出现。在大熊猫这样一个多配制物种,这个峰值可以看作与挑战假说相关联。在冬季,肾上腺皮质激素水平逐月升高,12月份出现峰值。在繁殖季节来临前,肾上腺皮质激素可能扮演了一个预备的角色。     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

Serum steroid hormones, sex hormone-binding globulin concentrations, and urinary hydroxylated estrogen metabolites in post-menopausal women in relation to daidzein-metabolizing phenotypes

Equol and O-desmethylangolensin (O-DMA) are products of bacterial metabolism of daidzein, an isoflavone in soybeans; thus, the presence or absence of equol and/or O-DMA in urine is a marker of particular intestinal bacteria profiles. Plasma hormone concentrations may be lower in pre-menopausal women who harbor the bacteria capable of producing equol (equol producers) compared to women who do not (equol non-producers). We evaluated concentrations of serum hormones, sex hormone-binding globulin (SHBG), and urinary 2-hydroxyestrone (2-OH E₁) and 16-hydroxyestrone (16-OH E₁) in relation to equol-producer and O-DMA-producer phenotypes in 89 post-menopausal women. Follicle stimulating hormone (FSH) was 23% greater in O-DMA-producers compared to non-producers (P=0.04). No significant differences in serum estrogens, androgens, metabolic hormones, or SHBG were observed in relation to either daidzein-metabolizing phenotype. Compared with non-producers within each phenotype, age-adjusted 2-OH E₁:16-OH E₁ was 27% greater (P=0.06) in equol-producers and 9% greater (P>0.10) in O-DMA-producers, and 2-OH E₁ concentrations were 24% greater in equol producers (P=0.07) and 42% greater in O-DMA producers (P=0.02). No significant differences in 16-OH E₁ were observed in relation to either phenotype. These results suggest that interindividual variability in intestinal bacteria may be related to differences in products of hormone metabolism in post-menopausal women.     

Steroid pathway and oestrone sulphate production in canine inflammatory mammary carcinoma

Spontaneous canine mammary inflammatory carcinoma (IMC) shares epidemiologic, histopathologic and clinical characteristics with the inflammatory breast carcinoma (IBC) disease in humans. We have analysed the steroids levels in serum and in tissue homogenates of IMC, the expression of two of their receptors (androgen and β-estrogen) and of three enzymes included in the steroidogenesis pathway (aromatase (CYP19A1), steroid sulphatase (STS) and estrogen sulfotransferase (EST)) trying to explain the specific accumulation of steroids in IMC tissues generating deposits in the form of lipid droplets whose presence can be attributed to steroids secreted by IMC cells. According to our working hypothesis, oestrone sulphate would be the main component of these lipid droplets. The presence of these steroid deposits would contribute to the intense proliferation and invasive behaviour of IMC and IBC, although their involvement in angiogenesis is yet to be demonstrated.     

Immunomagnetic capture of lens membrane fractions containing steroid binding protein

This study describes the use of magnetic Dynabeads to purify microsomes from a crude microsomal fraction. A 28 kDa membrane-associated protein is proposed to mediate the binding of progesterone and other steroid hormones to ocular lens membranes and the rapid-nongenomic actions of these steroids. The subcellular location of this membrane steroid binding protein (MSBP) was probed by capture of organelles containing MSBP by magnetic beads displaying an antibody to a cytoplasmic domain of the protein. The beads were exposed to a crude microsomal fraction from lens epithelia. Western blotting was used to identify captured organelles and confirm the presence of MSBP. Microsomes and trace fiber cell plasma membrane were captured. Microsomes contained the 28 kDa MSBP. Lens fiber cell membrane contained a 55 kDa immunoreactive protein. The role of this serendipitously recognized protein in binding of steroids is unknown.     

Steroid hormone signalling system and fungi

Three components of the steroid hormone signalling system, 17β-hydroxysteroid dehydrogenase, androgen binding proteins and steroid hormone signalling molecule testosterone were determined in the filamentous fungus Cochliobolus lunatus for the first time in a fungus. Their possible role in C. lunatus is discussed in comparison with their role in mammalian steroid hormone signalling system. The results are in accordance with the hypothesis, that the elements of the primordial signal transduction system should exist in present day eukaryotic microorganisms.     

Environmental physiology of the teleostean thyroid gland: a review

Synopsis Recent studies of thyroid hormone function are reviewed as they relate to the environmental physiology of teleost fish. In addition, reports dealing with the apparent interdependence of thyroid gland function with that of other endocrine glands are discussed with emphasis on the interrelated endocrine response associated with changing physiological status of teleosts.Seasonal changes in thyroid gland activity are described in several species. Although seasonal alterations in apparent thyroid status are concomitant with changes in ambient temperature, photoperiod and/or gonadal status, their biological significance is not fully understood and direct relationships are for the most part, not proven. Similarly, most reports of thyroid involvement in gonadal development or maturation are based on indirect evidence of the relationship. The exception to this is a study in immature or hypophysectomized goldfish in which thyroxine (T₄) was shown to promote ovarian development and maturation, possibly acting collateralistically or synergistically with gonadotropin. Even in this study it is not clear whether the T₄ effect is a direct action on the ovarian tissue or an indirect action via the regulation of metabolites necessary for gonad metabolism. Integumentary silvering and retinal porphyropsin formation in salmonids are stimulated by administration of T₄ or thyroid extracts. Administration of T₄ or triiodothyronine (T₃) enhances skeletal and somatic growth in some teleostean species, although the effect on somatic growth is most pronounced when these hormones act synergistically with somatotropin (STH) or androgens. The growth-promoting effects of T₄ and T₃ may be linked to their apparent involvement in lipid, carbohydrate, protein and vitamin metabolism. alterations in apparent thyroid activity concomitant with changes in ambient temperature have been reported (for example correlated with seasonal ambient thermal changes), although there are marked contradictions in data presented by different investigators. Reported temperature-related effects on thyroid function are probably secondary responses of thyroid metabolism to altered temperatures. Evidence of a direct rate of thyroid hormones in the regulation of migration (and associated behavioural modifications), salmonid smoltification, oxygen consumption, and osmotic or ionic regulation although highly suggestive in a number of areas is inconclusive and requires further critical experimental evaluation.The pituitary control (by thyrotropin) of thyroid secretion of T₄ is convincingly shown in several teleosts, and evidence of an inhibitory hypothalamic control of thyrotrop activity is highly suggestive in some species. A thyrotropic effect of somatotropin preparations is well established in several teleostean species; the effect does not appear to be related to contamination of the somatotropin preparations with thyrotropin, and may be an important consideration in explaining the apparently related involvement of T₄ (or T₃) and somatotropin in growth and metabolism. The apparent thyrotropic property of some gonadotropin preparations, shown in several teleostean species, requires further investigation before the doubts regarding hormone preparation purity can be satisfied. Recent studies of effects of prolactin on thyroid function are highly suggestive of an inhibitory role of prolactin in peripheral monodeiodination of T₄ to T₃ which secondarily affects thyroid activity in some species. There is no evidence of a direct involvement of corticotropin, melanotropin or fractions of these molecules on thyroid function in teleosts. Moreover, the little evidence in support of a role of gonadal or adrenocortical steroids in thyroid control is either often contradictory or indirect and needs to be evaluated further.Interlake epizootiological studies of thyroid dysfunction in Great Lakes salmonids provide substantive evidence for the presence of a ubiquitous waterborne goitrogen(s) in the Great Lakes environs. The nature of the goitrogen(s), whether naturally-occurring or a man-introduced toxicant, remains to be determined but the possible existence of waterborne goitrogens in natural water systems and their possible effects on experimental studies of teleostean thyroid function have to be evaluated further. If goitrogens are a common component of aquatic environments their presence could explain some of the data discrepancy among different groups of investigators, and could account for some of the apparent seasonal change in teleost thyroid physiology.     

Hormonal and experiential control of female-male mounting in the female rat

Mounting behavior in the female rat has been studied extensively in same-sex interactions, but not in the heterosexual dyad. The present study examined the display of female mounting of castrated noncopulating male rats (FMM). Ovariectomized (OVX) sexually naive female rats (N = 80) were given either estrogen (E) + progesterone (P), E + oil (O), P + O, or O + O treatment for five tests with castrated male rats. FMMs were observed in both the E + P and E + O females. The influence of the ovarian cycle on FMM was also investigated. Vaginal smears from sexually naive females (N = 16) were taken daily for 12 days immediately after testing with castrated males. FMM frequency was greatest during Proestrus. Finally, OVX females (N = 30) treated with E + P were given either 0, 1, 10 multi-ejaculatory heterosexual experiences with intact, sexually experienced males, prior to tests with intact, copulating or castrated, noncopulating males for five tests. Sexually naive females displayed a greater number of mounts relative to the sexually experienced females when tested with castrated, noncopulating males. In contrast, very few FMMs were observed in females of any group tested with intact, copulating males. These data suggest that FMM occurs naturally in rats as a "super-solicitational" behavior that is modified by hormone treatment and prior heterosexual experience.     

非哺乳类脊椎动物性别决定的研究进展

比较了非哺乳类脊椎动物和哺乳动物性别决定的差异,从影响非哺乳类脊椎动物性别决定的环境因子及其机制两方面回顾了性别决定的研究进展,分析环境因素和类固醇激素在性别决定中的作用,指出了非哺乳类脊椎动物性别决定研究中需要进一步探讨的问题。     

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target,serum-sensitive cells

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.