Calcium signaling in osteoclasts

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca²⁺) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca²⁺ oscillations occur during RANKL-mediated osteoclastogenesis. Ca²⁺ oscillations provide a digital Ca²⁺ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca²⁺/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca²⁺ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca²⁺ oscillations, and we discuss possible molecular players in this specialized Ca²⁺ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Localized calcium signaling in multinucleated osteoclasts

Localized intracellular Ca²⁺ ([Ca²⁺]_i) pulses, fluctuations, and repetitive spikes were detected in multinucleated rabbit osteoclasts in the presence of serum and in response to calcitonin using the fluorescent calcium indicator fluo-3 and a laser scanning microscope. We observed that these [Ca²⁺]_i changes were often restricted within a region of the cell body or propagated from the initial region of occurrence to other parts of the cell body but not to all parts. These observations suggest the existence of significant barriers to Ca²⁺ transport between different cytoplasmic regions of the osteoclast. To further investigate this phenomenon, we mechanically perturbed different cellular regions by touching locally with a micropipette. This usually induced a local increase in cytosolic and nuclear free [Ca²⁺]_i. In some cases there was propagation of the [Ca²⁺]_i increase to other regions but with part of the cell body not affected. Those regions of the cell body to which the [Ca²⁺]_i increase did not propagate had a [Ca²⁺]_i response to a direct mechanical perturbation. Our data show that osteoclasts can have different [Ca²⁺]_i activities in apparently equivalent cellular regions, no matter how generated. This suggests that there can be a number of spatially separate Ca²⁺ regulatory systems within an osteoclast cell body. © 1996 Wiley-Liss, Inc.     

Mechanisms of ATP-induced Ca signaling in osteoclasts

We invetigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-β-S abolishes the response in Ca²⁺-free medium, demonstrating an internal release of Ca²⁺ via a pathway that involves a G protein. GDP-β-S does not block in normal Ca²⁺-containing medium, suggesting that ATP also induces a Ca²⁺ influx across the cell membrane. We confirmed this using the Mn²⁺ quenching technique, which shows significant opening of Ca²⁺ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to β, γ-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our resutls indicate that the receptor is a P₂ subtype that is not characteristic of any previously reported P₂ receptor or combination of P₂ receptors.     

Regulation of podosomes by intracellular pH in avian osteoclasts

The effects of changes in intracellular pH (pH1) on the organization of the clear zone of isolated avian osteoclasts in culture were studied. The distribution of podosomes, the close contact areas that mediate the adhesion of osteoclasts to the substrate, was investigated by decoration of microfilaments with fluorescent phalloidin. Intracellular acidification by butyric acid induces significant increase of podosome formation at the level of the clear zone compared to controls. Conversely, alkalinization by HCO3- reduces the percentage of osteoclasts with podosomes. A role of pH1 on the adhesion of the osteoclasts to the substrate is hypothesized.     

Reactive oxygen species mediate RANK signaling in osteoclasts

RANKL, a member of tumor necrosis factor (TNF) superfamily, regulates the differentiation, activation, and survival of osteoclasts through binding to its cognate receptor, RANK. RANK can interact with several TNF-receptor-associated factors (TRAFs) and activates signaling molecules including Akt, NF-kappaB, and MAPKs. Although the transient elevation of reactive oxygen species (ROS) by receptor activation has been shown to act as a cellular secondary messenger, the involvement of ROS in RANK signaling pathways has been not characterized. In this study, we found that RANKL stimulated ROS generation in osteoclasts. Pretreatment of osteoclasts with the antioxidants N-acetyl-l-cystein and glutathione reduced RANKL-induced Akt, NF-kappaB, and ERK activation. The reduced NF-kappaB activity by antioxidants was associated with decreased IKK activity and IkappaBalpha phosphorylation. In contrast, antioxidants did not prevent TNF-alpha-induced Akt and NF-kappaB activation. Pretreatment with antioxidants also significantly reduced RANKL-induced actin ring formation, required for bone resorbing activity, and osteoclast survival. Taken together, our results suggest that ROS act as mediators in RANKL-induced signaling pathways and cellular events.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

Sphingosine-1-phosphate signaling controlling osteoclasts and bone homeostasis

Bone is a dynamic organ that is continuously turned over during growth, even in adults. During bone remodeling, homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, in pathological conditions such as osteoporosis, osteopetrosis, arthritic joint destruction, and bone metastasis, this equilibrium is disrupted. Since osteoclasts are excessively activated in osteolytic diseases, the inhibition of osteoclast function has been a major therapeutic strategy. It has recently been demonstrated that sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. By means of intravital multiphoton imaging of bone tissues, we have recently revealed that the reciprocal action of S1P controls the migration of osteoclast precursors between bone tissues and blood stream. Imaging technologies have enabled us to visualize the in situ behaviors of different cell types in intact tissues. In this review we also discuss future perspectives on this new method in the field of bone biology and medical sciences in general. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Dose-to-duration encoding and signaling beyond saturation in intracellular signaling networks

The cellular response elicited by an environmental cue typically varies with the strength of the stimulus. For example, in the yeast Saccharomyces cerevisiae, the concentration of mating pheromone determines whether cells undergo vegetative growth, chemotropic growth, or mating. This implies that the signaling pathways responsible for detecting the stimulus and initiating a response must transmit quantitative information about the intensity of the signal. Our previous experimental results suggest that yeast encode pheromone concentration as the duration of the transmitted signal. Here we use mathematical modeling to analyze possible biochemical mechanisms for performing this “dose-to-duration” conversion. We demonstrate that modulation of signal duration increases the range of stimulus concentrations for which dose-dependent responses are possible; this increased dynamic range produces the counterintuitive result of “signaling beyond saturation” in which dose-dependent responses are still possible after apparent saturation of the receptors. We propose a mechanism for dose-to-duration encoding in the yeast pheromone pathway that is consistent with current experimental observations. Most previous investigations of information processing by signaling pathways have focused on amplitude encoding without considering temporal aspects of signal transduction. Here we demonstrate that dose-to-duration encoding provides cells with an alternative mechanism for processing and transmitting quantitative information about their surrounding environment. The ability of signaling pathways to convert stimulus strength into signal duration results directly from the nonlinear nature of these systems and emphasizes the importance of considering the dynamic properties of signaling pathways when characterizing their behavior. Understanding how signaling pathways encode and transmit quantitative information about the external environment will not only deepen our understanding of these systems but also provide insight into how to reestablish proper function of pathways that have become dysregulated by disease.     

Regulatory roles and molecular signaling of TNF family members in osteoclasts

The tumor necrosis factor (TNF) family has been one of the most intensively studied families of proteins in the past two decades. The TNF family constitutes 19 members that mediate diverse biological functions in a variety of cellular systems. The TNF family members regulate cellular functions through binding to membrane-bound receptors belonging to the TNF receptor (TNFR) family. Members of the TNFR family lack intrinsic kinase activity and thus they initiate signaling by interacting intracellular signaling molecules such as TNFR associated factor (TRAF), TNFR associated death domain (TRADD) and Fas-associated death domain (FADD). In bone metabolism, it has been shown that numerous TNF family members including receptor activator of nuclear factor kappaB ligand (RANKL), TNF-alpha, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) play pivotal roles in the differentiation, function, survival and/or apoptosis of osteoclasts, the principal bone-resorbing cells. These TNF family members not only regulate physiological bone remodeling but they are also implicated in the pathogenesis of various bone diseases such as osteoporosis and bone loss in inflammatory conditions. This review will focus on our current understanding of the regulatory roles and molecular signaling of these TNF family members in osteoclasts.     

Revisiting intracellular calcium signaling semantics

Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.     

On sensitivity amplification in intracellular signaling cascades

Sensitivity amplification has long been regarded as a virtually universal property of signal transduction cascades, yet a comprehensive parameter analysis remains a challenge even for relatively simple networks. We use a fast and accurate method to compute properties of multilevel cascades of activation-inactivation cycles and show that the monocyclic cascades amplify sensitivity only under specific conditions. In particular, it is found that efficient sensitivity amplification in a cascade, relative to the sensitivities of individual cycles, requires asymmetry in saturation of converter enzymes, with inhibitors much more saturated than activators.     

Criticality in intracellular calcium signaling in cardiac myocytes

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.     

胞内钙信号作用的神经模型

细胞溶质内的游离钙离子在许多细胞活动中发挥着重要的作用.对于神经元,细胞膜上的神经电信号和胞内钙离子化学信号之间有着复杂的相互作用,每个神经元都可看作为一个含有细胞膜和内质网膜的双膜系统,而神经细胞的内质网则可视为神经元内的神经元.本综述探讨了神经元膜上神经电信号与内质网钙通道释放的胞内钙信号相耦合的动力学模型.我们认...     

Endothelial activation: intracellular signaling pathways

Tumor necrosis factor (TNF) is the prototypic proinflammatory cytokine and endothelial cells are the principal cellular targets of its actions. Here I review the responses of endothelial cells to TNF, with emphasis on the induction of endothelial leukocyte adhesion molecules. I focus on the biochemistry and cell biology of signal transduction in TNF-treated endothelial cells that lead to the expression of adhesion molecules.