Parathyroid hormone-activated volume-sensitive calcium influx pathways in mechanically loaded osteocytes

This paper documents for the first time a volume-sensitive Ca(2+) influx pathway in osteocytes, which transmits loading-induced signals into bone formation. Stretch loading by swelling rat and chicken osteocytes in hypo-osmotic solution induced a rapid and progressive increase of cytosolic calcium concentration, [Ca(2+)](i). The influx of extracellular Ca(2+) explains the increased [Ca(2+)](i) that paralleled the increase in the mean cell volume. Gadolinium chloride (Gd(3+)), an inhibitor of stretch- activated cation channels, blocked the [Ca(2+)](i) increase caused by hypotonic solutions. Also, the expression of alpha1C subunit of voltage-operated L-type Ca(2+) channels (alpha1C) is required for the hypotonicity-induced [Ca(2+)](i) increase judging from the effect of alpha1C antisense oligodeoxynucleotides. Parathyroid hormone (PTH) specifically potentiated the hypotonicity-induced [Ca(2+)](i) increase in a dose-dependent manner through the activation of adenyl cyclase. The increases induced by both PTH and hypotonicity were observed primarily in the processes of the osteocytes. In cyclically stretched osteocytes on flexible-bottomed plates, PTH also synergistically elevated the insulin-like growth factor-1 mRNA level. Furthermore, Gd(3+) and alpha1C antisense significantly inhibited the stretch-induced insulin-like growth factor-1 mRNA elevation. The volume-sensitive calcium influx pathways of osteocytes represent a mechanism by which PTH potentiates mechanical responsiveness, an important aspect of bone formation.     

佛手苷内酯对磷酸三钙磨损颗粒诱导骨细胞损伤的影响及机制*

目的:研究佛手苷内酯(BP)对磷酸三钙(TCP)磨损颗粒诱导骨细胞损伤的影响,并阐明其可能作用机制。方法:将TCP磨损颗粒与小鼠骨细胞MLO-Y4细胞共孵育48 h建立骨细胞体外损伤模型,随机分为正常对照(Control)组、TCP磨损颗粒(TCP,0.1 mg/ml)组、佛手苷内酯(1 μmol/L)组、佛手苷内酯(5 μmol/L)组和佛手苷内酯(20 μmol/L)组。MTT法和Calcein-AM染色检测各组骨细胞活性和形态改变;Hoechst 33342染色和流式细胞术分析各组骨细胞凋亡情况;实时荧光定量PCR检测各组骨细胞特征蛋白牙本质基质蛋白-1(DMP-1)、骨硬化蛋白(SOST)、成纤维细胞生长因子23(FGF23)的mRNA水平;Western blot法检测各组骨细胞中内质网应激标志蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核细胞翻译起始因子2α (eIF2α)、磷酸化eIF2α(p-eIF2α)、活性转录因子(ATF4)和 C/EBP 同源蛋白(CHOP)等的表达及caspase-3的活化变化。结果:与Control组比较,TCP组骨细胞的活性和DMP-1的mRNA水平显著降低(P＜0.05),骨细胞凋亡率及SOST、FGF23的mRNA水平显著增加(P＜0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/eIF2α值显著升高;与TCP组比较,佛手苷内酯组骨细胞损伤明显减轻,骨细胞凋亡率显著减少(P＜ 0.05),GRP78、ATF4和CHOP等蛋白质表达、p-PERK/PERK值和p-eIF2α/PERK值也明显下降(P＜0.05)。结论:佛手苷内酯可明显抑制TCP磨损颗粒所致的骨细胞损伤,其机制可能与减弱TCP磨损颗粒诱导的内质网应激反应及PERK通路的活化密切相关。     

Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO-Y4 osteocytes

Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain-induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJs) between osteocytes and osteoblasts provides a mechanism whereby flow-induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and prostaglandin E(2) (PGE(2)) increases in response to flow. Unapposed connexons, or hemichannels, are themselves functional and may constitute a novel mechanotransduction mechanism. Using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes, we examined the time course and mechanism of hemichannel activation in response to fluid flow, the composition of the hemichannels, and the role of hemichannels in flow-induced ATP release. We demonstrate that fluid flow activates hemichannels in MLO-Y4, but not MC3T3-E1, through a mechanism involving protein kinase C, which induces ATP and PGE(2) release.     

Expression profile of Notch-1 in mechanically overloaded plantaris muscle of mice

AimWe investigated the expression pattern of Notch-1 in normal and hypertrophied plantaris muscle of mice.Main methodsWe performed immunofluorescence of both Notch-1 and the Notch-1-linking molecules.Key findingsImmunofluorescence labeling revealed Notch-1 protein in Pax7-positive satellite cells during days 2–6. We observed clear co-localization between Notch-1 and myogenin (4.9 ± 1.3%) in the hypertrophied muscle at 4 days. Several mononuclei (possibly satellite cells) possessed both Notch-1 and Foxo1 in the plantaris muscle subjected to mechanical overloading (4.1 ± 1.2%).SignificanceNotch-1 may play an important role in the maintenance of quiescent satellite cells.     

Corticosterone regulates the expression of neuropeptide Y and reelin in MLO-Y4 cells

Osteocytes that have a dendritic appearance are widely believed to form a complex cellular network system and play crucial roles in mechanotransduction as a principal bone mechanosensor, which is the basis of their neuronallike biology, as previously reported. Neuropeptide Y (NPY) and reelin mRNA, which are brain-specific neurogenic markers, have been identified in osteocytes. However, changes in the production of NPY and reelin in response to specific biochemical stimulation are unknown. In this study, we investigated the in vitro effect of corticosterone, one of the endogenous glucocorticoids, on the expression of NPY and reelin in the MLO-Y4 osteocyte cell line. Cells were treated with corticosterone at different concentrations (10(-9) M-10(-5) M) for 1, 3, 6, 12 and 24 h. As revealed, corticosterone reduced the MLO-Y4 cell viability and proliferation in a dose- and time-dependent manner based on an MTT assay and a Vi-CELL analyzer. The cells were then incubated with corticosterone (10(-6) μM), and the NPY and reelin expression levels were detected at 1, 3, 6, 12 and 24 h using real-time PCR and Western blot analysis. These results demonstrated that at the gene and the protein levels, corticosterone significantly upregulated the NPY and reelin expression in a time-dependent manner. The application of a glucocorticoid receptor antagonist, RU486, reversed the reduced cell viability and the increased expression of NPY and reelin that were caused by corticosterone. To the best of our knowledge, this is the first report to verify that corticosterone regulates the NPY and reelin expression in osteocytes.     

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.     

Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon.

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.     

Skeletal muscle morphology in power-lifters with and without anabolic steroids

The morphological appearance of the vastus lateralis (VL) muscle from high-level power-lifters on long-term anabolic steroid supplementation (PAS) and power-lifters never taking anabolic steroids (P) was compared. The effects of long- and short-term supplementation were compared. Enzyme-immunohistochemical investigations were performed to assess muscle fiber type composition, fiber area, number of myonuclei per fiber, internal myonuclei, myonuclear domains and proportion of satellite cells. The PAS group had larger type I, IIA, IIAB and IIC fiber areas (p<0.05). The number of myonuclei/fiber and the proportion of central nuclei were significantly higher in the PAS group (p<0.05). Similar results were seen in the trapezius muscle (T) but additionally, in T the proportion of fibers expressing developmental myosin isoforms was higher in the PAS group compared to the P group. Further, in VL, the PAS group had significantly larger nuclear domains in fibers containing ≥5 myonuclei. The results of AS on VL morphology in this study were similar to previously reported short-term effects of AS on VL. The initial effects from AS appear to be maintained for several years.     

二维回转培养对MLO-Y4骨样细胞PKD2表达定位 及胞内钙信号的影响

目的：PKD2(polycystin2,多囊肾病蛋白2)能够在细胞膜上形成无选择性的阳离子通道,在肾上皮细胞中PKD2 与初级纤毛 共定位,通过改变胞内的钙信号过程参与细胞对力学刺激的响应。本实验通过二维回转培养来模拟失重效应,旨在探讨二维回转 培养对MLO-Y4 骨样细胞PKD2 表达定位,及胞内钙信号的影响。初步了解PKD2 在小鼠骨样细胞MLO-Y4 响应力学刺激过程 中起的作用。方法：采用二维回转培养骨样细胞MLO-Y4,用RT-PCR和western blotting检测PKD2的表达,用荧光共聚焦显微镜 检测细胞中PKD2 与初级纤毛的定位及细胞内钙离子含量。结果：与对照组相比,在二维回转培养后,骨样细胞MLO-Y4 的PKD2 表达在mRNA和蛋白水平都有明显的下降,PKD2、PKD1(polycystin1,多囊肾病蛋白1)和乙酰化的α-tubulin 共定位,同时二维回 转培养降低了细胞内钙离子含量。结论：在二维回转培养下,PKD2可能通过调节自身表达来改变细胞膜上PKD 通道的数目和开 放情况来影响细胞内钙离子含量,参与骨细胞对细胞外应力的感受过程,其详细机制还有待进一步实验研究。这将对探讨骨细胞 响应力学刺激的具体机制提供重要的理论依据。     

Two-dimensional clinorotation influences cellular morphology, cytoskeleton and secretion of MLO-Y4 osteocyte-like cells

The aim of this study was to investigate the effects of the clinostat-simulated weightlessness on biological characteristics of MLO-Y4 osteocyte-like cells. MLO-Y4 cells were incubated for 24 h, then randomly divided into 3 groups and rotated in a clinostat as a model of simulated weightlessness for 12 h, 24 h and 48 h. The morphology, cytoskeleton, and secretion of soluble molecules of MLO-Y4 cells were observed and detected. The results show that clinostat culture affects the number of dendrites/cell, cytoskeleton distribution, and secretion of nitric oxide and prostaglandin E2 in MLO-Y4 cells. These results may provide some clue to explore the cellular mechanism of bone loss caused by weightlessness.     

Mechanical behavior of an individual adherent MLO-Y4 osteocyte under shear flow

Mechanical properties of a single cell and its mechanical response under stimulation play an important role in regulating interactions between cell and extracellular matrix and affecting mechanotransduction. Osteocytes exhibit solid-like viscoelastic behavior in response to the interstitial fluid shear resulting from tissue matrix deformation. This study intends to quantitatively describe the mechanical behavior of osteocytes combining in vitro experiment and fluid–structure interaction (FSI) finite element (FE) model. The cell is configured in the FSI FE model using the observed data from quasi-3D images. Instead of simply assigning the cellular viscoelastic parameters by statistical data, the mechanical parameters are determined by an iterative algorithm comparing the experimental and the computational results from the FE model. The viscoelastic parameters of osteocytes are obtained as: the equilibrium elasticity modulus \(k_{1}=0.15\pm 0.038\,\hbox {kPa}\), instantaneous elasticity modulus \((k_{1}+k_{2})=0.77\pm 0.23\,\hbox {kPa}\), viscosity coefficient \(\eta =1.38\pm 0.33\,\hbox {kPa}\,\hbox {s}\). A novel index to quantify the cell adhesion is also put forward. In addition, an interesting competition phenomenon is revealed on the cell surface concerning stress and strain, i.e., the place with high stress has low strain and that with low stress has high strain. The proposed method provides a novel technique to study the mechanical behavior of individual adherent cell in vitro. It is believed that this quantitative technique not only determines cell mechanical behavior but also helps elucidate the mechanism of mechanotransduction in various types of cells.     

Effect of anabolic steroids on rat heart muscle cells

Summary Long-term treatment of female rats with the anabolic steroid hormone Methandrostenolone results in a conspicuous increase of intermediate sized, nonmyofibrillar filaments in muscle cells of the left cardiac ventricle, as revealed by electron microscopy. These filaments, measuring 70–110 Å in diameter, form a characteristic network at the Z-level of the sarcomere, either encircling or penetrating the Z-bands, and appear to insert into the nuclear membrane. The T-system is accompanied by the filaments adjacent to the site of the couplings. Here they are attached to subsarcolemmal electron-dense patches, which may be Z-line precursor material. The filaments may function as a cytoskeleton, to provide passive support in the mechanism of contraction and to mediate nucleo-sarcolemmal and nucleomyofibrillar exchange.The author wishes to thank Prof. Dr. C. Stang-Voss for advice and discussion     

Expression of Glut-4 and Glut-1 transporters in rat diaphragm muscle

Glucose transporters (Gluts) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. Generally, alterations of Gluts expression in limb skeletal muscle have been reported. However, the changes of Glut isoforms in respiratory muscle which contracts with a duty cycle have rarely been studied. This study was performed to evaluate at the light microscopy level the expression of Glut-4 and Glut-1 transporters in normal and denervated diaphragm by immunohistochemistry method with specific Gluts antibodies. The results showed Glut-4 immunoreactivity in both the cell periphery and the interior of myocytes. Glut-1 was also present in the cell border and in the interior of myocytes in control diaphragm. However, Glut-4 staining was stronger than Glut-1 staining in control diaphragm. In denervated hemidiaphragm, the Glut-4 immunolabelling decreased and Glut-1 increased. These data indicated that (1) Glut-4 and Glut-1 transporters were observed in diaphragm; and (2) there were alterations in the expression of both glucose transporters after denervation. These alterations in Glut isoforms after denervation may be associated with the removal of innervation itself, and/or may partly result from passive stretch imposed by inspiratory activation of the contralateral side.     

Effect of muscle action and metabolic strain on oxidative metabolic responses in human skeletal muscle.

A recent report suggests that differences in aerobic capacity exist between concentric and eccentric muscle action in human muscle (T. W. Ryschon, M. D. Fowler, R. E. Wysong, A. R. Anthony, and R. S. Balaban. J. Appl. Physiol. 83: 867-874, 1997). This study compared oxidative response, in the form of phosphocreatine (PCr) resynthesis rates, with matched levels of metabolic strain (i.e., changes in ADP concentration or the free energy of ATP hydrolysis) in tibialis anterior muscle exercised with either muscle action in vivo (n = 7 subjects). Exercise was controlled and metabolic strain measured by a dynamometer and (31)P-magnetic resonance spectroscopy, respectively. Metabolic strain was varied to bring cytosolic ADP concentration up to 55 microM or decrease the free energy of ATP hydrolysis to -55 kJ/mol with no change in cytoplasmic pH. PCr resynthesis rates after exercise ranged from 31.9 to 462.5 and from 21.4 to 405.4 micromol PCr/s for concentric and eccentric action, respectively. PCr resynthesis rates as a function of metabolic strain were not significantly different between muscle actions (P > 0.40), suggesting that oxidative capacity is dependent on metabolic strain, not muscle action. Pooled data were found to more closely conform to previous biochemical measurements when a term for increasing oxidative capacity with metabolic strain was added to models of respiratory control.     

Metabolic history and metabolic fitness as drivers of anabolic heterogeneity in isogenic microbial populations

Microbial populations often display different degrees of heterogeneity in their substrate assimilation, that is, anabolic heterogeneity. It has been shown that nutrient limitations are a relevant trigger for this behaviour. Here we explore the dynamics of anabolic heterogeneity under nutrient replete conditions. We applied time-resolved stable isotope probing and nanoscale secondary ion mass spectrometry to quantify substrate assimilation by individual cells of Pseudomonas putida, P. stutzeri and Thauera aromatica. Acetate and benzoate at different concentrations were used as substrates. Anabolic heterogeneity was quantified by the cumulative differentiation tendency index. We observed two major, opposing trends of anabolic heterogeneity over time. Most often, microbial populations started as highly heterogeneous, with heterogeneity decreasing by various degrees over time. The second, less frequently observed trend, saw microbial populations starting at low or very low heterogeneity, and remaining largely stable over time. We explain these trends as an interplay of metabolic history (e.g. former growth substrate or other nutrient limitations) and metabolic fitness (i.e. the fine-tuning of metabolic pathways to process a defined growth substrate). Our results offer a new viewpoint on the intra-population functional diversification often encountered in the environment, and suggests that some microbial populations may be intrinsically heterogeneous.     

Effect of anabolic steroids on overloaded and overloaded suspended skeletal muscle

The purpose of this study was to ascertain whether anabolic steroids act synergistically with functional overload in terms of increasing muscle weight and subcellular protein content of normal overloaded and suspended overloaded rodent plantaris muscle. Female rats were randomly assigned to six groups (7 rats/group) for 6 wk: 1) normal control (NC), 2) overload (OV), 3) overload steroid (OV-S), 4) normal suspended (N-SUS), 5) overload suspended (OV-SUS), and 6) overload suspended steroid (OV-SUS-S). Rats receiving anabolic steroid were administered 0.3 mg nandrolone decanoate (Deca-Durabolin) per day. Relative to control values, overload induced 1) sparing of muscle weight of the OV-SUS group as well as larger absolute and normalized (mg muscle/g body wt) muscle weight of the OV group (P less than 0.05), 2) greater protein content (mg/muscle, P less than 0.05), and 3) an increased relative expression of slow myosin in both the OV and OV-SUS groups (P less than 0.05). Although anabolic steroid treatment of overload animals (OV-S) did not alter further the pattern of response of any parameter analyzed for the OV group, it did induce larger absolute and normalized muscle weight (P less than 0.05) as well as a greater protein content (mg/muscle, P less than 0.05) of the OV-SUS-S group compared with control values. However, anabolic steroid treatment did not alter the pattern of isomyosin expression observed in the overload (OV-S vs. OV) or overload suspended (OV-SUS-S vs. OV-SUS) groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

PTH receptors and apoptosis in osteocytes

Osteocytes comprise a heterogenous population of terminally differentiated osteoblasts that direct bone remodeling in response to applied mechanical loading of bone. Increased osteocyte density accompanies the anabolic effect of PTH in vivo, whereas accelerated osteocyte death may be precipitated by estrogen deficiency or excess glucocorticoid exposure (conditions benefitted by intermittent PTH therapy) and by renal failure (where circulating intact PTH and, especially, PTH carboxylfragments are elevated). Osteocytes express type-1 PTH/ PTHrP receptors (PTH1Rs), which are fully activated by aminoterminal PTH fragments and couple to multiple signal transducers, including adenylyl cyclase and phospholipase C. Activation of PTH1Rs in osteocytes promotes gap junction-mediated intercellular coupling, increases expression of MMP-9, potentiates calcium influx via stretch-activated cation channels, amplifies the osteogenic response to mechanical loading in vivo, and regulates apoptosis. Control of osteocyte apoptosis by PTH1Rs is complex, in that intermittent PTH(1-34) administration reduces the fraction of vertebral apoptotic osteocytes at 1 month in adult mice but increases femoral metaphyseal osteocyte apoptosis at 1-2 weeks in young rats. In MLO-Y4 cells, PTH(1-34) prevents apoptosis otherwise induced within 6 hr by dexamethasone. In older studies, large doses of intact PTH(1-84) caused rapid "degenerative" morphologic changes in osteocytes, similar to those described in renal osteodystrophy. We isolated clonal conditionally immortalized osteocytic (OC) cell lines from mice homozygous for targeted ablation of the PTH1R gene. OC cells express abundant (2-3 x 10(6) per cell) receptors specific for the carboxyl(C)-terminus of intact PTH(1-84) ("CPTHRs") but, as expected, do not express PTH1Rs or respond to PTH(1-34). CPTHRs are expressed at much lower levels by other skeletally-derived cell lines. Several highly conserved ligand determinants of CPTHR binding have been identified, including PTH(24-27), PTH(53-54) and the sequence PTH(55-84), loss of which reduces binding affinity by over 100-fold. Human PTH(53-84), like PTH(1-84), PTH(24-84), and PTH(39-84), increases OC cell apoptosis. Ala-scanning mutagenesis to define sequences within PTH(55-84) important for binding and bioactivity is underway. We conclude that osteocytes may be important targets for CPTH fragments that are secreted by the parathyroid glands or generated by peripheral metabolism of intact PTH and that accumulate in blood, especially in renal failure. Studies of functional interplay between responses to CPTHRs and (transfected) PTH1Rs, using receptor-specific ligands in OC cells, should provide new insight into PTH regulation of osteocyte function and survival.