Estrogen inhibits osteoclasts formation and bone resorption via microRNA-27a targeting PPARγ and APC

Inhibition of osteoclasts formation and bone resorption by estrogen is very important in the etiology of postmenopausal osteoporosis. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell differentiation and proliferation. Thus, we hypothesized that these regulatory molecules might be implicated in the process of estrogen-decreased osteoclasts formation and bone resorption. Western blot, quantitative real-time polymerase chain reaction, tartrate-resistant acid phosphatase staining, pit formation assay and luciferase assay were used to investigate the role of microRNAs in estrogen-inhibited osteoclast differentiation and bone resorption. We found that estrogen could directly suppress receptor activator of nuclear factor B ligand/macrophage colony-stimulating factor-induced differentiation of bone marrow-derived macrophages into osteoclasts in the absence of stromal cell. MicroRNA-27a was significantly increased during the process of estrogen-decreased osteoclast differentiation. Overexpressing of microRNA-27a remarkably enhanced the inhibitory effect of estrogen on osteoclast differentiation and bone resorption, whereas which were alleviated by microRNA-27a depletion. Mechanistic studies showed that microRNA-27a inhibited peroxisome proliferator-activated receptor gamma (PPARγ) and adenomatous polyposis coli (APC) expression in osteoclasts through a microRNA-27a binding site within the 3′-untranslational region of PPARγ and APC. PPARγ and APC respectively contributed to microRNA-27a-decreased osteoclast differentiation and bone resorption. Taken together, these results showed that microRNA-27a may play a significant role in the process of estrogen-inhibited osteoclast differentiation and function.     

Enantiomeric separation of aminophosphonic acids by capillary electrophoresis

Racemic aminophosphonic acids were completely resolved into their enantiomers by capillary electrophoresis using β-cyclodextrin as a chiral selector in a borate electrolyte. The reproducibility of sample injection, solute migration time, and detection limits of the solute were studied. The calibration curve obtained from peak areas was linear over the concentration range of 10 to 300 μg/mL. © 1996 Wiley-Liss, Inc.     

AAV‐mediated expression of secreted and transmembrane αKlotho isoforms rescues relevant aging hallmarks in senescent SAMP8 mice

Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting a decrease in the organism''s protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction, and worsening of physical status. In this context, we explored the AAV9‐mediated expression of the two main isoforms of the aging‐protective factor Klotho (KL) as a strategy to prevent these general age‐related features using the senescence‐accelerated mouse prone 8 (SAMP8) model. Both secreted and transmembrane KL isoforms improved cognitive performance, physical state parameters, and different molecular variables associated with aging. Epigenetic landscape was recovered for the analyzed global markers DNA methylation (5‐mC), hydroxymethylation (5‐hmC), and restoration occurred in the acetylation levels of H3 and H4. Gene expression of pro‐ and anti‐inflammatory mediators in central nervous system such as TNF‐α and IL‐10, respectively, had improved levels, which were comparable to the senescence‐accelerated‐mouse resistant 1 (SAMR1) healthy control. Additionally, this improvement in neuroinflammation was supported by changes in the histological markers Iba1, GFAP, and SA β‐gal. Furthermore, bone tissue structural variables, especially altered during senescence, recovered in SAMP8 mice to SAMR1 control values after treatment with both KL isoforms. This work presents evidence of the beneficial pleiotropic role of Klotho as an anti‐aging therapy as well as new specific functions of the KL isoforms for the epigenetic regulation and aged bone structure alteration in an aging mouse model.     

Oxidative damage increases with age in a canine model of human brain aging

We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and beta-amyloid (Abeta) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Abeta. Abeta was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Abeta, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Abeta deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

Advances in the discovery of cathepsin K inhibitors on bone resorption

Cathepsin K (Cat K), highly expressed in osteoclasts, is a cysteine protease member of the cathepsin lysosomal protease family and has been of increasing interest as a target of medicinal chemistry efforts for its role in bone matrix degradation. Inhibition of the Cat K enzyme reduces bone resorption and thus, has rendered the enzyme as an attractive target for anti-resorptive osteoporosis therapy. Over the past decades, considerable efforts have been made to design and develop highly potent, excellently selective and orally applicable Cat K inhibitors. These inhibitors are derived from synthetic compounds or natural products, some of which have passed preclinical studies and are presently in clinical trials at different stages of advancement. In this review, we briefly summarised the historic development of Cat K inhibitors and discussed the relationship between structures of inhibitors and active sites in Cat K for the purpose of guiding future development of inhibitors.     

小鼠衰老对不同组织中亚精胺的影响

目的:本研究以模式小鼠C57BL为对象,研究小鼠在衰老过程中不同组织器官内源性亚精胺含量的变化。方法:利用高效液相色谱检测小鼠心脏和肝脏组织中亚精胺含量,进一步应用qRT-PCR以及Western blot检测在衰老过程中,不同组织器官中亚精胺生物合成途径的关键基因表达变化,利用亚精胺处理细胞检测DNA损伤应答能力。结果:随着衰老的发生心脏(199.09±17.12)和肝脏组织(168.92±5.12)中亚精胺含量显著降低,分别为78.01±13.52、62.05±6.73,差异有统计学意义(P0.05);不同组织器官中亚精胺生物合成途径的关键基因Odc、Srm、Amd1的表达随衰老的发生明显下调,并且伴随着DNA损伤应答障碍;利用亚精胺处理细胞,能够增强细胞对DNA损伤的应答反应。结论:衰老的小鼠中内源性亚精胺含量降低,并且其合成途径的关键基因转录水平降低,导致细胞对DNA损伤应答能力减弱,从而加速机体衰老进程。     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.