Function of Estrogen on Bone and the Characterization of the Skeletal Phenotype of Steroid Receptor Coactivator (SRC)-1 KO Mice

Estrogen is known to have important effects on both reproductive and non-reproductive tissues. In this study it was demonstrated that an E2 dose of as little as 5µg/kg/d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), and it had no stimulatory effects on the uterus in 6 month old C57BL/6 mice. By contrast, when 3 month old C56BL/6 mice were administered the same doses of E2 and studied after 1 month; the 5 g/kg/d dose resulted in uterine hypertrophy, but was not able to prevent loss of cancellous bone. These results, thus, a) provide data on the dose response for E2 effects on mouse bone; and b) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential ?selective? effects on bone versus reproductive tissues. 2. Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen action in a number of tissues. The presented study has established that SRC-1 KO female and male mice have a comparable skeletal phenotype to their WT littermates at 3 and 5 months of age. The treatment of ovariectomized SRC-1 KO female mice with a physiological concentration of E2 led to a predominant defect in estrogen action in cancellous bone, with a relative preservation of estrogen effects on cortical bone. However, the deficit in estrogen action in the female SRC-1 KO mice was overcome by using a higher dose of E2, consistent with estrogen resistance in bone. The differential expression of the interacting nuclear receptors, ER- and -, in cancellous versus cortical bone and the specific interactions of these receptor isoforms with SRC-1 may, in part, explain why cancellous bone is more susceptible to loss of SRC-1 than cortical bone. 3. In contrast, the SRC-1 male mice lack the defect in estrogen action on bone. These findings are consistent with a gender-related difference that in male mice ER-α but not ER- is mediating the estrogen action on bone. The treatment of SRC-1 KO male mice with 5α-DHT resulted in a significant better response of the cortical bone from the KO male mice compared to the WT mice. The findings lead to the suggestion that in male WT mice SRC-1 might inhibit the action of 5α-DHT on bone. 4. Peripheral tissues including bone are dependent on circulating active sex steroids, but also synthesize estrogen from circulating C19 precursor locally. The conversion of the precursors to estrogen is mediated by aromatase. In this work it has been shown that human and rodent osteoblastic cells at different stages of differentiation express aromatase. Furthermore, the expression and activity has been demonstrated in bone tissues of mice and rats. Although a regulation of the aromatase by E2 was reported by other investigators in certain tissues, such as breast tissue and mammary glands, I did not find a change of aromatase expression and activity in the various bone cell lines and bone tissue under estrogen deficiency and estrogen exposure

Function of Estrogen on Bone and the Characterization of the Skeletal Phenotype of Steroid Receptor Coactivator (SRC)-1 KO Mice

Estrogen is known to have important effects on both reproductive and non-reproductive tissues. In this study it was demonstrated that an E2 dose of as little as 5µg/kg/d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), and it had no stimulatory effects on the uterus in 6 month old C57BL/6 mice. By contrast, when 3 month old C56BL/6 mice were administered the same doses of E2 and studied after 1 month; the 5 g/kg/d dose resulted in uterine hypertrophy, but was not able to prevent loss of cancellous bone. These results, thus, a) provide data on the dose response for E2 effects on mouse bone; and b) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential ?selective? effects on bone versus reproductive tissues. 2. Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen action in a number of tissues. The presented study has established that SRC-1 KO female and male mice have a comparable skeletal phenotype to their WT littermates at 3 and 5 months of age. The treatment of ovariectomized SRC-1 KO female mice with a physiological concentration of E2 led to a predominant defect in estrogen action in cancellous bone, with a relative preservation of estrogen effects on cortical bone. However, the deficit in estrogen action in the female SRC-1 KO mice was overcome by using a higher dose of E2, consistent with estrogen resistance in bone. The differential expression of the interacting nuclear receptors, ER- and -, in cancellous versus cortical bone and the specific interactions of these receptor isoforms with SRC-1 may, in part, explain why cancellous bone is more susceptible to loss of SRC-1 than cortical bone. 3. In contrast, the SRC-1 male mice lack the defect in estrogen action on bone. These findings are consistent with a gender-related difference that in male mice ER-α but not ER- is mediating the estrogen action on bone. The treatment of SRC-1 KO male mice with 5α-DHT resulted in a significant better response of the cortical bone from the KO male mice compared to the WT mice. The findings lead to the suggestion that in male WT mice SRC-1 might inhibit the action of 5α-DHT on bone. 4. Peripheral tissues including bone are dependent on circulating active sex steroids, but also synthesize estrogen from circulating C19 precursor locally. The conversion of the precursors to estrogen is mediated by aromatase. In this work it has been shown that human and rodent osteoblastic cells at different stages of differentiation express aromatase. Furthermore, the expression and activity has been demonstrated in bone tissues of mice and rats. Although a regulation of the aromatase by E2 was reported by other investigators in certain tissues, such as breast tissue and mammary glands, I did not find a change of aromatase expression and activity in the various bone cell lines and bone tissue under estrogen deficiency and estrogen exposure

Function of Estrogen on Bone and the Characterization of the Skeletal Phenotype of Steroid Receptor Coactivator (SRC)-1 KO Mice

Estrogen is known to have important effects on both reproductive and non-reproductive tissues. In this study it was demonstrated that an E2 dose of as little as 5µg/kg/d completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), and it had no stimulatory effects on the uterus in 6 month old C57BL/6 mice. By contrast, when 3 month old C56BL/6 mice were administered the same doses of E2 and studied after 1 month; the 5 g/kg/d dose resulted in uterine hypertrophy, but was not able to prevent loss of cancellous bone. These results, thus, a) provide data on the dose response for E2 effects on mouse bone; and b) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential ?selective? effects on bone versus reproductive tissues. 2. Steroid receptor coactivator (SRC)-1 is an important nuclear receptor coactivator that enhances estrogen action in a number of tissues. The presented study has established that SRC-1 KO female and male mice have a comparable skeletal phenotype to their WT littermates at 3 and 5 months of age. The treatment of ovariectomized SRC-1 KO female mice with a physiological concentration of E2 led to a predominant defect in estrogen action in cancellous bone, with a relative preservation of estrogen effects on cortical bone. However, the deficit in estrogen action in the female SRC-1 KO mice was overcome by using a higher dose of E2, consistent with estrogen resistance in bone. The differential expression of the interacting nuclear receptors, ER- and -, in cancellous versus cortical bone and the specific interactions of these receptor isoforms with SRC-1 may, in part, explain why cancellous bone is more susceptible to loss of SRC-1 than cortical bone. 3. In contrast, the SRC-1 male mice lack the defect in estrogen action on bone. These findings are consistent with a gender-related difference that in male mice ER-α but not ER- is mediating the estrogen action on bone. The treatment of SRC-1 KO male mice with 5α-DHT resulted in a significant better response of the cortical bone from the KO male mice compared to the WT mice. The findings lead to the suggestion that in male WT mice SRC-1 might inhibit the action of 5α-DHT on bone. 4. Peripheral tissues including bone are dependent on circulating active sex steroids, but also synthesize estrogen from circulating C19 precursor locally. The conversion of the precursors to estrogen is mediated by aromatase. In this work it has been shown that human and rodent osteoblastic cells at different stages of differentiation express aromatase. Furthermore, the expression and activity has been demonstrated in bone tissues of mice and rats. Although a regulation of the aromatase by E2 was reported by other investigators in certain tissues, such as breast tissue and mammary glands, I did not find a change of aromatase expression and activity in the various bone cell lines and bone tissue under estrogen deficiency and estrogen exposure

Analysis of the interaction of influenza virus polymerase complex with human cell factors

12 pages, 4 figures.-- PMID: 18491320 [PubMed].-- Supplementary information (Suppl. figure S1, 2 pages) available at: http://www.wiley-vch.de/contents/jc_2120/2008/pro200700508_s.pdfThe influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.N. J. was a fellow from Ministerio de Educación y Ciencia. E. T. was a fellow from Instituto de Salud Carlos III. P. G. was a fellow from Gobierno Vasco. This work was supported by the Spanish Ministry of Education and Science (Ministerio de Educación y Ciencia) (grant BFU2004-491), the VIRHOST Program financed by Comunidad de Madrid, European Vigilance Network for the Management of Antiviral Drug Resistance (VIRGIL) and the FLUPOL strep project (SP5B-CT-2007-044263).Peer reviewe

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies. © 2012 Coronnello et al

Relation of Age, Gender, and Bone Mass to Circulating Sclerostin Levels in Women and Men

Sclerostin is a potent inhibitor of Wnt signaling and bone formation. However, there is currently no information on the relation of circulating sclerostin levels to age, gender, or bone mass in humans. Thus we measured serum sclerostin levels in a population-based sample of 362 women [123 premenopausal, 152 postmenopausal not on estrogen treatment (ET), and 87 postmenopausal on ET] and 318 men, aged 21 to 97 years. Sclerostin levels (mean ± SEM) were significantly higher in men than women (33.3 ± 1.0 pmol/L versus 23.7 ± 0.6 pmol/L, p < .001). In pre- and postmenopausal women not on ET combined (n = 275) as well as in men, sclerostin levels were positively associated with age (r = 0.52 and r = 0.64, respectively, p < .001 for both). Over life, serum sclerostin levels increased by 2.4- and 4.6-fold in the women and men, respectively. Moreover, for a given total-body bone mineral content, elderly subjects (age ≥ 60 years) had higher serum sclerostin levels than younger subjects (ages 20 to 39 years). Our data thus demonstrate that (1) men have higher serum sclerostin levels than women, (2) serum sclerostin levels increase markedly with age, and (3) compared with younger subjects, elderly individuals have higher serum sclerostin levels for a given amount of bone mass. Further studies are needed to define the cause of the age-related increase in serum sclerostin levels in humans as well as the potential role of this increase in mediating the known age-related impairment in bone formation. © 2011 American Society for Bone and Mineral Research