HuR-positive stress granules: Potential targets for age-related osteoporosis

Aging impairs osteoblast function and bone turnover, resulting in age-related bone degeneration. Stress granules (SGs) are membrane-less organelles that assemble in response to stress via the recruitment of RNA-binding proteins (RBPs), and have emerged as a novel mechanism in age-related diseases. Here, we identified HuR as a bone-related RBP that aggregated into SGs and facilitated osteogenesis during aging. HuR-positive SG formation increased during osteoblast differentiation, and HuR overexpression mitigated the reduction in SG formation observed in senescent osteoblasts. Moreover, HuR positively regulated the mRNA stability and expression of its target β-catenin by binding and recruiting β-catenin into SGs. As a potential therapeutic target, HuR activator apigenin (API) enhanced its expression and thus aided osteoblasts differentiation. API treatment increased HuR nuclear export, enhanced the recruitment of β-catenin into HuR-positive SGs, facilitated β-catenin nuclear translocation, and contributed osteogenesis. Our findings highlight the roles of HuR and its SGs in promoting osteogenesis during skeletal aging and lay the groundwork for novel therapeutic strategies against age-related skeletal disorders.     

有氧运动联合褪黑素对Ⅱ型糖尿病大鼠骨质疏松的影响

目的：观察有氧运动和褪黑素对Ⅱ型糖尿病大鼠骨质疏松的影响。方法：6周龄的成年雌性SD大鼠60只,随机分为安静对照组（N组）10只和Ⅱ型糖尿病模型组50只,N组大鼠不加任何干预,Ⅱ型糖尿病模型组大鼠一次性腹腔注射35 mg/kg链脲佐菌素（STZ）,1周后检测大鼠血糖大于16.7 mmol/L为Ⅱ型糖尿病造模成功,将40只成模大鼠随机分为糖尿病对照组（D）、糖尿病+有氧运动组（DE）、糖尿病+褪黑素组（DM）、糖尿病+有氧运动+褪黑素组（DEM）,每组10只;DE组和DEM组大鼠采用20 min的递增负荷的方式进行跑台有氧运动,训练持续6周,DM组和DEM组大鼠每天灌胃40 mg/kg褪黑素,观察各组大鼠体重、脊椎骨以及左右股骨骨密度（BMD）、观察大鼠血糖、血清丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、血清总钙（Ca）、无机磷（P）和甲状旁腺素（PTH）的变化。结果：与N组相比,D组大鼠体重、血清SOD、GSH-Px水平、血Ca、腰椎和左右股骨BMD显著降低（P < 0.05,P < 0.01）,血糖、血清MDA和血PTH水平显著升高（P < 0.01）,血P无明显变化（P > 0.05）;与D组比较,DE组、DM组大鼠大鼠体重、血清SOD、GSH-Px水平、血Ca、腰椎和左右股骨BMD显著升高（P < 0.05,P <0.01）,血糖、血清MDA和血PTH水平显著降低（P < 0.05,P < 0.01）,血P无明显变化（P > 0.05）,有氧运动和褪黑素同时干预效果更好。结论：有氧运动和褪黑素均能改善糖尿病骨质疏松,且两者联合干预的效果更加显著,其可能与通过提高糖尿病大鼠的抗氧化应激能力,调节糖的代谢从而有效地降低血钙和PTH,改善BMD来缓解骨质疏松有关。     

骨质疏松症易感基因BDNF的遗传学关联分析及功能研究

为探索脑源性神经营养因子BDNF基因与中国汉族人群骨密度(bone mineral density, BMD)的关系,解析该基因调节骨质疏松症的功能机制,进而为中国汉族人群的骨质疏松症的防治提供依据。本研究从我国陕西地区征集了1300例汉族样本,并测量髋部/脊椎骨密度。选取BDNF基因上的14个标签SNPs进行基因分型,与1300例样本BMD进行关联分析,发现8个SNPs与髋部/脊椎BMD显著关联(P < 0.05)。其中,SNP rs16917237同时与髋部和脊椎骨密度关联,经Bonferroni校正后仍表现出显著性(0.05/14 = 0.0036)。整合连锁不平衡和单体型分析、表观功能注释、表达数量基因座分析、代谢通路分析进一步探索BDNF基因调节骨质疏松症的机制。构建小鼠前成骨细胞系(MC3T3-E1)人骨形态形成蛋白(rhBMP-2)诱导分化模型,利用小干扰RNA(siRNA)敲除BDNF。结果显示：14个SNPs位于同一单体型内;rs16917237在成骨细胞中表现出较强的激活型组蛋白H3K4me1、H3K4me3、H3K27ac修饰信号以及P300结合信号,表明其在成骨细胞中可能具有调控活性;rs16917237在11个组织中均能显著影响BDNF基因表达;BDNF基因位于与成骨细胞增殖分化相关的MAPK通路中;BDNF敲低能够显著降低MAPK通路中与成骨分化相关的CREB 基因mRNA和蛋白表达水平,提示其可能通过调控CREB表达进而影响成骨分化。生物信息学分析和功能实验结果一致,表明BDNF基因可能是影响骨质疏松症的重要功能基因。     

Is HMGB1 an osteocyte alarmin?

The death of osteocytes, the terminally differentiated cells of the osteoblast lineage that are embedded in bone and regulate remodeling, is significant to both normal and pathological bone resorption. Apoptotic osteocytes putatively release a clarion signal that enhances the development of the bone-resorbing osteoclasts and targets their migration to the breach in the osteocyte network. This phenomenon is thought to underlie normal repair of bone microdamage and contribute to the etiologies of inflammatory bone loss. The chromatin protein high mobility group box 1 protein (HMGB1) has been identified as an "alarmin" in other tissues. An alarmin is an endogenous molecule released by dead and dying cells that alert the innate immune system to damage and the need for tissue repair. Wang and colleagues presented evidence in a landmark 1999 study showing that released HMGB1 is a lethal mediator of sepsis. Extracellular HMGB1 is a ligand for the toll-like receptors (TLRs) and for the receptor for advanced glycation end products (RAGE) all of which amplify inflammation. Recent studies by our lab and others have shown that HMGB1 is a bone-active cytokine. It is released by apoptotic osteoblasts in vitro, including the MLO-Y4 osteocyte-like cells. Extracellular HMGB1 enhances the expression of RANKL, TNFalpha, and IL6 in osteoblastogenic bone marrow stromal cell cultures, and it is chemotactic to osteoclasts. In this prospectus we will review HMGB1 activity at the immune-bone interface and propose a role for HMGB1 as an osteocyte alarmin and mediator of normal remodeling and inflammatory bone loss.     

老年性痴呆症患者海马老年斑三维重构

老年斑是老年性痴呆 (AD)的主要病理特征之一。β 淀粉样蛋白 (Aβ)在老年斑中的积聚与AD发病关系密切。因此 ,如何抑制老年斑内Aβ的积聚和促进Aβ纤维的解聚对于治疗和预防AD有十分重要的意义。通过免疫组织化学染色、MATLAB软件图像处理、AVS重构等技术构建了老年斑三维结构。实验发现海马部位的弥散斑和经典斑具有孔状结构 ;弥散斑无明显核心结构 ,密度分布趋势由中心向周围递减 ;经典斑中心Aβ沉积最致密 ,周围Aβ沉积最少 ,而斑块外周Aβ沉积密度居中。老年斑三维结构重构方法的建立 ,有助于老年斑积聚动力学的研究 ,并对AD病理机制的探讨具有重要意义。     

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1–0.4 g at 30 Hz for 10–60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM‐induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM‐induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse‐induced bone loss. J. Cell. Biochem. 106: 306–316, 2009. © 2009 Wiley‐Liss, Inc.     

Predicting changes in mechanical properties of trabecular bone by adaptive remodeling

Because changes in the mechanical properties of bone are closely related to trabecular bone remodeling, methods that consider the temporal morphological changes induced by adaptive remodeling of trabecular bone are needed to estimate long-term fracture risk and bone quality in osteoporosis. We simulated bone remodeling using simplified and pig trabecular bone models and estimated the morphology of healthy and osteoporotic cases. We then displayed the fracture risk of the remodeled models based on a cumulative histogram from high stress. The histogram showed more elements had higher stresses in the osteoporosis model, indicating that the osteoporosis model had a greater risk.     

DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age

Age‐related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age‐dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1‐Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP‐Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP‐Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21^CIP1 levels, as well as increased levels of GATA4 and activation of NF‐κB – two major stimulators of the senescence‐associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro‐osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation.