PERK signaling pathway in bone metabolism: Friend or foe?

Osteoblasts and osteoclasts participate in the process of bone remodelling to meet the needs of normal growth and development or repair pathological damage. Endoplasmic reticulum stress (ER stress) can break the intracellular homeostasis of osteoclasts and osteoblasts, which is closely related to abnormal bone remodelling. The double‐stranded RNA‐dependent protein kinase (PKR)‐like ER kinase (PERK) is a key transmembrane protein that regulates ER stress, and growing evidence suggests that the PERK pathway plays a crucial role in regulating bone metabolism under both physiological and pathological conditions. Based on the current findings, we summarized the main mechanisms involved in bone metabolism downstream of the PERK pathway, among which elF2α, FOXO1, CaN, Nrf2 and DAG play a role in regulating the differentiation of osteoblasts and osteoclasts. Importantly, strategies by the regulation of PERK pathway exert beneficial effects in preclinical trials of several bone‐related diseases. Given the importance and novelty of PERK pathway, we provide an overview and discuss the roles of PERK pathway in regulating bone metabolism and its impact on bone‐related diseases. We hope that the development of research in this field will bring a bright future for the treatment of bone‐related diseases.

Effects of downstream changes caused by activation of PERK on osteoblastogenesis and osteoclastogenesis. Phosphorylated elF2a, Nrf2, DAG, FOXO1 and enhanced activity of CaN have different impacts on osteogenic and osteoclast differentiation, respectively. Green squares indicate the expression of the genes.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

Free fatty acid receptor 4-β-arrestin 2 pathway mediates the effects of different classes of unsaturated fatty acids in osteoclasts and osteoblasts

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω−3 and ω−7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, β-arrestin 2 (βarr2) and G_αq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω−3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω−7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/βarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω−6 UFA, arachidonic acid, and the ω−9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/βarr2 signalling axis. This study reveals that FFAR4/βarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.     

Calcium Signalling and Alzheimer’s Disease

New insights into how Ca²⁺ regulates learning and memory have begun to provide clues as to how the amyloid-dependent remodelling of neuronal Ca²⁺ signalling pathways can disrupt the mechanisms of learning and memory in Alzheimer’s disease (AD). The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway remodels the neuronal Ca²⁺ signalling pathways responsible for cognition by enhancing the entry of Ca²⁺ and/or the release of internal Ca²⁺ by ryanodine receptors or InsP₃ receptors. The specific proposal is that Ca²⁺ signalling remodelling results in a persistent elevation in the level of Ca²⁺ that constantly erases newly acquired memories by enhancing the mechanism of long-term depression (LTD). Neurons can still form memories through the process of LTP, but this stored information is rapidly removed by the persistent activation of LTD. Further dysregulation in Ca²⁺ signalling will then go on to induce the neurodegeneration that characterizes the later stages of dementia.     

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.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

The calcium/NFAT pathway: role in development and function of regulatory T cells

Calcium signals are essential for diverse cellular functions in the immune system. Sustained Ca²⁺ entry is necessary for complete and long-lasting activation of calcineurin/NFAT pathways. A growing number of studies have emphasized that Ca²⁺/calcineurin/NFAT pathway is crucial for both development and function of regulatory T cells.     

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca²⁺ signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca²⁺ was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca²⁺] and environmental temperatures. Therefore, Ca²⁺ signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.     

Acidic Ca(2+) stores come to the fore

Changes in the concentration of cytosolic Ca²⁺ form the basis of a ubiquitous signal transduction pathway. Accumulating evidence implicates acidic organelles in the control of Ca²⁺ dynamics in organisms across phyla. In this special issue, we discuss Ca²⁺ signalling by these “acidic Ca²⁺ stores” which include acidocalcisomes, vacuoles, the endo-lysosomal system, lysosome-related organelles, secretory vesicles and the Golgi complex. Ca²⁺ release from these morphologically very different organelles is mediated by members of the TRP channel superfamily and two-pore channels. Inositol trisphosphate and ryanodine receptors which are traditionally viewed as endoplasmic reticulum Ca²⁺ release channels can also mobilize acidic Ca²⁺ stores. Ca²⁺ uptake into acidic Ca²⁺ stores is driven by Ca²⁺ ATPases and Ca²⁺/H⁺ exchangers. In animal cells, the Ca²⁺-mobilizing messenger NAADP plays a central role in mediating Ca²⁺ signals from acidic Ca²⁺ stores through activation of two-pore channels. These signals are important for several physiological processes including muscle contraction and differentiation. Dysfunctional acidic Ca²⁺ stores have been implicated in diseases such as acute pancreatitis and lysosomal storage disorders. Acidic Ca²⁺ stores are therefore emerging as essential components of the Ca²⁺ signalling network and merit extensive further study.     

How calcium signals in myocytes and pericytes are integrated across in situ microvascular networks and control microvascular tone

The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca²⁺ signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca²⁺ signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca²⁺ signalling and contractility of myocytes and pericytes. Ca²⁺ signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca²⁺ signals arise from Ca²⁺ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca²⁺ release and not via ryanodine receptors or Ca²⁺ entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca²⁺ signals between arteriolar and venular networks dependent on gap junctions and Ca²⁺ entry via L-type Ca²⁺ channels. Endothelial Ca²⁺ signalling inhibits intracellular Ca²⁺ oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca²⁺ signals via EDHF. Increases of Ca²⁺ in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes.     

Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress

Mechanical stimuli can improve bone function by promoting the proliferation and differentiation of bone cells and osteoblasts. As precursors of osteoblasts, human mesenchymal stem cells (hMSCs) are sensitive to mechanical stimuli. In recent years, fluid shear stress (FSS) has been widely used as a method of mechanical stimulation in bone tissue engineering to induce the osteogenic differentiation of hMSCs. However, the mechanism of this differentiation is not completely clear. Several signaling pathways are involved in the mechanotransduction of hMSCs responding to FSS, such as MAPK, NO/cGMP/PKG and Ca²⁺ signaling pathway. Here, we briefly review how hMSCs respond to fluid flow stimuli and focus on the signal molecules involved in this mechanotransduction.     

Effects of electromagnetic stimulation on the functional responsiveness of isolated rat osteoclasts

We report the effects of pulsed electromagnetic fields (PEMFs) on the responsiveness of osteoclasts to cellular, hormonal, and ionic signals. Osteoclasts isolated from neonatal rat long bones were dispersed onto either slices of devitalised cortical bone (for the measurement of resorptive activity) or glass coverslips (for the determination of the cytosolic free Ca²⁺ concentration, [Ca²⁺]). Osteoclasts were also cocultured on bone with osteoblastlike, UMR-106 cells. Bone resorption was quantitated by scanning electron microscopy and computer-assisted morphometry. PEMF application to osteoblast–osteoclast cocultures for 18 hr resulted in a twofold stimulation of bone resorption. In contrast, resorption by isolated osteoclasts remained unchanged in the presence of PEMFs, suggesting that osteoblasts were necessary for the PEMF-induced resorption simulation seen in osteoblast–osteoclast cocultures. Furthermore, the potent inhibitory action of the hormone calcitonin on bone resorption was unaffected by PEMF application. However, PEMFs completely reversed another quite distinct action of calcitonin on the osteoclast: its potent inhibitory effect on the activation of the divalent cation-sensing (or Ca²⁺) receptor. For these experiments, we made fura 2-based measurements of cytosolic [Ca²⁺] in single osteoclasts in response to the application of a known Ca²⁺ receptor agonist, Ni²⁺. We first confirmed that activation of the osteoclast Ca²⁺ receptor by Ni²⁺ (5 mM) resulted in a characteristic monophasic elevation of cytosolic [Ca²⁺]. As shown previously, this response was attenuated strongly by calcitonin at concentrations between 0.03 and 3 nM but remained intact in response to PEMFs. PEMF application, however, prevented the inhibitory effect of calcitonin on Ni²⁺-induced cytosolic Ca²⁺ elevation. This suggested that the fields disrupted the interaction between the calcitonin and Ca²⁺ receptor systems. In conclusion, we have shown that electromagnetic fields stimulate bone resorption through an action on the osteoblast and, by abolishing the inhibitory effects of calcitonin, also restore the responsiveness of osteoclasts to divalent cations. J. Cell. Physiol. 176:537–544, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.