Estrogen protects primary osteocytes against glucocorticoid-induced apoptosis

Glucocorticoid-induced osteoporosis may be at least in part due to the increased apoptosis of osteocytes. To study the role of osteocyte apoptosis in glucocorticoid-induced osteoporosis, we isolated primary osteocytes from murine calvaria for the analysis of the effects of dexamethasone in in vitro culture. The cells were identified by morphology, cytochemical staining, immunocytochemical staining and mRNA expression of phosphate-regulating gene with homology to endopeptidases on the X chromosome (PHEX) and sclerosteosis/van Buchem disease gene (SOST). We found that dexamethasone induced osteocyte apoptosis in a dose-dependent manner. A glucocorticoid receptor antagonist, mifepristone (RU486), suppressed dexamethasone-induced osteocyte apoptosis, suggesting that it was mediated by glucocorticoid receptor. Immunocytochemical stainings showed that glucocorticoid receptors are present in primary osteocytes, and they were translocated to nuclei after the exposure to dexamethasone. Addition of estrogen prevented glucocorticoid receptor translocation into nuclei. Corresponding antiapoptotic effects in primary osteocytes were also seen after the pretreatment of primary osteocytes with a picomolar concentration of estrogen. The pure antiestrogen ICI 182,780 inhibited estrogen effect on apoptosis induced by dexamethasone. These data suggest that glucocorticoid receptors play an important role in glucocorticoid-induced osteocyte apoptosis. Most importantly, estrogen has a protective effect {against osteocyte}{ }{apoptosis}. To conclude, the mechanism of glucocorticoid-induced osteoporosis may be due to the apoptosis of osteocytes, which can be opposed by estrogen.     

SGK1 is essential for meiotic resumption in mammalian oocytes

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation.In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.     

Osteogenic growth peptide effects on primary human osteoblast cultures: potential relevance for the treatment of glucocorticoid-induced osteoporosis

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10-14), reproducing the OGP C-terminal active portion [H-Tyr-Gly-Phe-Gly-Gly-OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, have demonstrated that OGP (10-14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10-14) in clinical therapy, we have tested different concentrations of OGP (10-14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10-14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10-14) inhibits hOB apoptosis induced by an excess of dexamethasone (-48% of apoptotic cells). Furthermore, we show that OGP (10-14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10-14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC-induced osteoporosis.     

Alpha-crystallin is a target gene of the farnesoid X-activated receptor in human livers

Alpha-crystallins comprise 35% of soluble proteins in the ocular lens and possess chaperone-like functions. Furthermore, the alphaA subunit (alphaA-crystallin) of alpha crystallin is thought to be "lens-specific" as only very low levels of expression were detected in a few non-lenticular tissues. Here we report that human alphaA-crystallin is expressed in human livers and is regulated by farnesoid X-activated receptor (FXR) in response to FXR agonists. AlphaA-crystallin was identified in a microarray screen as one of the most highly induced genes after treatment of HepG2 cells with the synthetic FXR ligand GW4064. Northern blot and quantitative real-time PCR analyses confirmed that alphaA-crystallin expression was induced in HepG2-derived cell lines and human primary hepatocytes and hepatic stellate cells in response to either natural or synthetic FXR ligands. Transient transfection studies and electrophoretic mobility shift assays revealed a functional FXR response element located in intron 1 of the human alphaA-crystallin gene. Importantly, immunohistochemical staining of human liver sections showed increased alphaA-crystallin expression in cholangiocytes and hepatocytes. As a member of the small heat shock protein family possessing chaperone-like activity, alphaA-crystallin may be involved in protection of hepatocytes from the toxic effects of high concentrations of bile acids, as would occur in disease states such as cholestasis.     

Association of SGK1 gene polymorphisms with type 2 diabetes

The serum and glucocorticoid inducible kinase SGK1 is genomically upregulated by glucocorticoids and in turn stimulates a variety of carriers and channels including the renal epithelial Na(+) channel ENaC and the intestinal Na(+) glucose transporter SGLT1. Twin studies disclosed an association of a specific SGK1 haplotype with moderately enhanced blood pressure in individuals who are carrying simultaneously a homozygous genotype for a variant in intron 6 [I6CC] and a homozygous or heterozygous genotype for the C allele of a polymorphism in exon 8 [E8CC/CT] of the SGK1 gene. A subsequent study confirmed the impact of this risk haplotype on blood pressure. SGK1 knockout mice are resistant to the insulin and high salt induced increase of blood pressure, glucocorticoid induced increase of electrogenic glucose transport, and glucocorticoid induced suppression of insulin release. The present study explored whether the I6CC/E8CC/CT haplotype impacts on the prevalence of type 2 diabetes. The prevalence of the I6CC genotype was 3.1% in a healthy German, 2.4 % in a healthy Romanian and 11.6 % in a healthy African population from Ghana (p=0.0006 versus prevalence in Caucasians). Comparison of genotype frequencies between type 2 diabetic patients and the respective control groups revealed significant differences for the intron 6 T>C variant. Carriers of at least one T allele were protected against type 2 diabetes (Romanians: p=0.023; OR 0.29; 95% CI 0.09-0.89; Germans: p=0.01; OR 0.37; 95% CI 0.17-0.81). The SGK1 risk haplotype (I6CC/E8CC/CT) was significantly (p=0.032; OR 4.31, 95% CI 1.19-15.58) more frequent in diabetic patients (7.2 %) than in healthy volunteers from Romania (1.8%). The observations support the view that SGK-1 may participate in the pathogenesis of metabolic syndrome.     

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells’ progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral modiated gene transfer efficiency in primary T-lymphocytes, in particular T cell activation status, viral receptor expression, and culture conditions.     

Direction of movement is encoded in the human primary motor cortex

The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1.     

Normal and glucocorticoid-induced development of the human small intestinal xenograft

The aim of this study was to determine whether intestinal xenografts could recapitulate human in utero development by using disaccharidases as markers. Twenty-week-old fetal intestine was transplanted into immunocompromised mice and was followed. At 20-wk of gestation, the fetal human intestine was morphologically developed with high sucrase and trehalase but had low lactase activities. By 9-wk posttransplantation, jejunal xenografts were morphologically and functionally developed and were then monitored for 相似文献