Gender dependent effects of testosterone and 17β-estradiol on bone growth and modelling in young mice

This study examined the effects of estrogen (17β-estradiol) and testosterone on the growth of long bones in male and female mice, with and without gonadectomy. Weight and nose-to-tail length were determined at 3 weeks of age at time of gonadectomy, 7 days later at the onset of hormone therapy, and throughout the treatment period. Gonadectomized mice exhibited an initial weight gain during the pretreatment period but length was unaffected. Hormone treatment altered weight gain in surgical and intact animals in a gender- and hormone-dependent manner. Estradiol enhanced weight gain in intact mice, but inhibited weight gain in ovariectomized mice. Lower doses of estradiol increased weight gain in orchiectomized mice at early time points. Testosterone increased weight in intact females and males, but not in gonadectomized mice. Estradiol increased nose-to-tail length in intact females at early time points, but inhibited length in ovariectomized females at later times, and it decreased length in intact males. Testosterone increased length in normal females and normal males. Serum Ca was unaffected by ovariectomy, but orchiectomy resulted in decreased levels. Estradiol reduced serum Ca in gonadectomized animals; serum Ca was increased by estradiol treatment in intact females. Changes in tibial bone weight, ash weight and mineral composition, and relative sizes of epiphyseal and metaphyseal bone were gender-, gonadectomy- and hormone-specific. Bone weight was greater in ovariectomized mice. Ash weight per bone was comparable, but there was an increase in Ca and P content with ovariectomy. Estradiol increased bone weight, ash content, and bone Ca and P in ovariectomized and intact females. Orchiectomy alone did not alter bone weight, ash content, or Ca and P, but orchiectomized mice were sensitive to estradiol; all parameters were increased in the orchiectomized animals treated with estradiol. Analysis of the ash content and Ca and P per mg bone, rather than per bone, demonstrated estradiol and testosterone alter net bone formation, but not the amount of mineral per unit bone. Ovariectomy increased hypertrophic cartilage. While estradiol did not alter tibial area in ovariectomized mice, it caused an increase in intact females. The total amount of growth plate cartilage in ovariectomized animals was decreased by estradiol to levels typical of intact animals due to a greater decrease in the hypertrophic cartilage in the ovariectomized mice, as well as a greater increase in metaphyseal bone area. Testosterone had no effect on these parameters in the females. Orchiectomy decreased the amount of growth plate cartilage, but increased the hypertrophic zone. Estradiol increased growth plate cartilage in intact male mice, but decreased it in orchiectomized mice. This difference was also seen in the hypertrophic zone. Total growth plate cartilage and hypertrophic cartilage were increased by testosterone in intact males, whereas metaphyseal and epiphyseal bone area were decreased. The results show for the first time that there is a gender-specific response in both male and female mice to both estradiol and testosterone, whether or not the animals have been gonadectomized. For many parameters, orchiectomized mice behave like females in response to both sex steroids, indicating that the male gonad is needed for mouse bone to exhibit the male phenotypic response to estradiol and testosterone.     

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.     

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.     

Modeling of the effects of IL-17 and TNF-α on endothelial cells and thrombus growth

Rheumatoid and psoriatic arthritis are chronic inflammatory diseases, with massive increase of cardiovascular events (CVE), and contribution of the cytokines TNF-α and IL-17. Chronic inflammation inside the joint membrane or synovium results from the activation of fibroblasts/synoviocytes, and leads to the release of cytokines from monocytes (Tumor Necrosis Factor or TNF) and from T lymphocytes (Interleukin-17 or IL-17). At the systemic level, the very same cytokines affect endothelial cells and vessel wall. We have previously shown [1], [2] that IL-17 and TNF-α, specifically when combined, increase procoagulation, decrease anticoagulation and increase platelet aggregation, leading to thrombosis. These results are the basis for the models of interactions between IL-17 and TNF, and genes expressed by activated endothelial cells. This work is devoted to mathematical modeling and numerical simulations of blood coagulation and clot growth under the influence of IL-17 and TNF-α. We show that they can provoke thrombosis, leading to the complete or partial occlusion of blood vessels. The regimes of blood coagulation and conditions of occlusion are investigated in numerical simulations and in approximate analytical models. The results of mathematical modeling allow us to predict thrombosis development for an individual patient.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.     

The effects of polymorphisms in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels are dependent on environmental context

Thrombosis is a key factor in the pathophysiology of cardiovascular disease. Important biochemical constituents of the fibrinolytic system, affecting thrombosis, include tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both t-PA and PAI-1 are determined by multiple genetic and environmental factors. We aimed to investigate whether the effects of polymorphism in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on t-PA or PAI-1 levels are dependent on environmental factors in a large population-based sample from the PREVEND study in Groningen, The Netherlands (n = 2,527). We found strong evidence (P ≤ 0.02) for interaction effects of polymorphisms from the bradykinin receptor (BDKRB2) gene and alcohol consumption on t-PA in females and males and on PAI-1 in males. Only suggestive evidence (P ≤ 0.10) was present for an interaction effect of the BDKRB2 gene and alcohol consumption on PAI-1 levels in females. Another consistent finding was evidence for an interaction between bradykinin receptor (BDKRB2) gene polymorphisms and body size as measured by body mass index and/or waist–hip-ratio. For each gender and for both t-PA and PAI-1 there was at least one BDKRB2–body size combination that exhibited suggestive (P ≤ 0.10), significant (P ≤ 0.04) and/or strong evidence (P ≤ 0.02) for interaction. In conclusion, the genetic architecture of t-PA and PAI-1 is dependent on the environmental context such as body size and alcohol use. The present study emphasizes the importance of including environmental factors in genetic analyses to fully comprehend the genetic architecture of a specific trait. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential effects of interleukin-1 and transforming growth factor β on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers

Small proteoglycans (PGs) are supposed to play great roles in the assembly of cartilage matrix but the influence of cytokines and growth factors on their synthesis by articular chondrocytes is largely unknown. We investigated whether EL-1 and TGF1 influence the production of small leucine-rich proteoglycans by chondrocytes cultured in a three-dimensional gel, as compared to the common monolayer system.Rabbit articular chondrocytes were cultured in alginate beads for 14 days or as monolayers for 7 days. The effect of 2 ng/ml IIL-1 or TGF1 during the last two days in culture was determined, after [³⁵S]methionine labeling over the last 24 h. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment of alginate beads whereas medium and cell-layer fractions were isolated from monolayer cultures. Total newly synthesized PGs were first isolated by anion-exchange chromatography and the small PGs were further separated from aggrecans by gel-filtration (Sepharose CL-4B) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).Addition of TGF1 resulted in an overall rise in neosynthesized small PG content in both culture systems. However, TGF1 significantly increased to the same extent the percentage of small PGs laid down in the cell-associated and the further-removed matrix compartments of the beacls culture (+00%) whereas it auirnted the content of small PGs in the medium (+40%) and reduced that of the cell fraction (+35%) in the monolayer culture. By adding IL-1, the amount of total newly synthesized small PGs was decreased in monolayers while it increased in alginate beads. IL-1 was also shown to change the relative distribution of these molecules in the monolayer system in contrast to the alginate beads culture where the proportions were not significantly altered. Electrophoretic analyes of the ³⁵S-labeled small PGs-containing fractions confirmed these effects at the level of the 45-50 kDa-related core proteins.This study demonstrates that TGF and IL-1 differently influence small PG synthesis of rabbit articular chondrocytes depending on whether they are cultured in alginate beads or in monolayers. Moreover, the regulation of small PG expression appears to be different from that of high-molecular weight aggrecans. As these small molecules are playing major roles in matrix assembly and growth factor regulation, the data may have great relevance to the pathogenesis of osteoarthritis and repair of articular cartilage lesions.     

A combination of ascorbic acid and α-tocopherol or a combination of Mg and Zn are both able to reduce the adverse effects of lindane-poisoning on rat brain and liver

The purpose of this study, carried out on male Wistar rats, was to evaluate the beneficial effects of supplementation with ascorbic acid (Vit C) and α-tocopherol (Vit E) or with Mg and Zn upon lindane-induced damages in liver and brain. Under our experimental conditions, lindane poisoning (5 mg/kg body weight per day for 3 days) resulted in (1) an increased level of plasma glucose, cholesterol and triglycerides, (2) an increased activity of LDH, ALP, AST, ALT, (3) an oxidative stress in liver and brain as revealed by an increased level of lipids peroxidation (TBARS) and a decrease of glutathione-peroxidase, superoxide dismutase and catalase activities in liver and brain. In conclusion, both Vit C + E or Mg + Zn treatments display beneficial effects upon oxidative stress induced by lindane treatment in liver and brain.