Modulation Of Calcium Signaling Research Articles

Modulation of intracellular calcium signaling by microRNA-34a-5p

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca2+ influx through Ca2+ release-activated Ca2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions.

Atorvastatin ameliorates the contractile dysfunction of the aorta induced by organ culture.

Statins are widely used in the treatment of hypercholesterolemia. Studies have demonstrated that statins could maintain vascular contractile function through inhibiting the transformation of vascular smooth muscle cells (VSMCs) from the contractile phenotype to the synthetic phenotype. However, the underlying mechanisms have not been fully elucidated. The effect of atorvastatin on the thoracic aorta of Sprague-Dawley rats cultured in serum-free conditions in vitro was evaluated. Aortic constriction was induced by high potassium, phenylephrine, and CaCl2. The protein expression levels of α1 adrenoceptor; inositol 1,4,5-trisphosphate (IP3) receptor; protein kinase Cδ (PKCδ); stromal interaction molecule 1 (STIM1); high-voltage activated dihydropyridine-sensitive (L type, Cav1.2) channels; and two contractile phenotype marker proteins [α-smooth muscle actin (α-SMA) and myosin (SM-MHC)] were determined by western blotting. Compared with the fresh control, the constriction of rat aorta was impaired after culture in serum-free medium for 24h. The impaired contraction of cultured aortas was mediated by Cav1.2 and store-operated Ca2+ (SOC) channel, which could be improved by atorvastatin at 20μM. The protein expression levels of α1 adrenoceptor, IP3 receptor, PKCδ, STIM1, Cav1.2, α-SMA, and SM-MHC in the aortas cultured in serum-free conditions were decreased significantly. Atorvastatin partially prevented the reduction in the contractility and the downregulation of these proteins in cultured aortas. The transformation of the VSMC phenotype is associated with the vasoconstriction dysfunction of cultured aortas. Atorvastatin may protect vascular function by modulating calcium signaling pathways.

The KCa3.1 blocker TRAM-34 inhibits proliferation of fibroblasts in paraquat-induced pulmonary fibrosis

KCa3.1, a Ca2+-activated K+ channel, plays an important role in modulating calcium signaling and maintaining membrane potential during cell activation. It has been reported to promote fibroblast function in many fibrotic diseases. However, the role of KCa3.1 in the pathophysiology of pulmonary fibrosis after paraquat (PQ) poisoning has not been studied. A rat model of PQ poisoning was used. After treatment with TRAM-34, which is a highly selective KCa3.1 blocker, the expression of KCa3.1, TGF-β1 and α-SMA were evaluated via Western blot, histology and other assays. Bromodeoxyuridine (BrdU) marking and MTT assay were used to measure primary rat pulmonary fibroblast proliferation. The results showed that KCa3.1 expression was elevated after PQ poisoning. Blockade of KCa3.1 alleviated PQ-induced pulmonary inflammation and fibrosis. Blockade of KCa3.1 also attenuated the level of collagen I and α-SMA and the proliferation of fibroblasts. However, TGF-β1 expression remained unaffected by blockade of KCa3.1 in rat lung tissues after PQ poisoning. The present study suggests that KCa3.1 expression increased and might promote pulmonary fibroblast proliferation in PQ-induced pulmonary fibrosis. In addition, we confirmed that TRAM-34 attenuates proliferation and collagen secretion of fibroblasts. Our findings indicated that TRAM-34 might inhibit PQ-induced proliferation of pulmonary fibroblasts and prevent progression of lung fibrosis.

A Fluidic Culture Platform for Spatially Patterned Cell Growth, Differentiation, and Cocultures.

Stem cell cultures within perfusion bioreactors, while efficient in obtaining cell numbers, often lack the similarity to native tissues and consequently cell phenotype. We develop a three-dimensional (3D)-printed fluidic chamber for dynamic stem cell culture, with emphasis on control over flow and substrate curvature in a 3D environment, two physiologic features of native tissues. The chamber geometry, consisting of an array of vertical cylindrical pillars, facilitates actin-mediated localization of human mesenchymal stem cells (hMSCs) within ∼200 μm distance from the pillars, enabling spatial patterning of hMSCs and endothelial cells in cocultures and subsequent modulation of calcium signaling between these two essential cell types in the bone marrow microenvironment. Flow-enhanced osteogenic differentiation of hMSCs in growth media imposes spatial variations of alkaline phosphatase expression, which positively correlates with local shear stress. Proliferation of hMSCs is maintained within the chamber, exceeding the cell expansion in conventional static culture. The capability to manipulate cell spatial patterning, differentiation, and 3D tissue formation through geometry and flow demonstrates the culture chamber's relevant chemomechanical cues in stem cell microenvironments, thus providing an easy-to-implement tool to study interactions among substrate curvature, shear stress, and intracellular actin machinery in the tissue-engineered construct.

Gene expression profiles of immune-regulatory genes in whole blood of cattle with a subclinical infection of Mycobacterium avium subsp. paratuberculosis.

Johne’s disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), resulting in inflammation of intestines and persistent diarrhea. The initial host response against MAP infections is mainly regulated by the Th1 response, which is characterized by the production of IFN-γ. With the progression of disease, MAP can survive in the host through the evasion of the host’s immune response by manipulating the host immune response. However, the host response during subclinical phases has not been fully understood. Immune regulatory genes, including Th17-derived cytokines, interferon regulatory factors, and calcium signaling-associated genes, are hypothesized to play an important role during subclinical phases of Johne’s disease. Therefore, the present study was conducted to analyze the expression profiles of immune regulatory genes during MAP infection in whole blood. Different expression patterns of genes were identified depending on the infection stages. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.

Sphingolipid-mediated calcium signaling and its pathological effects

Metabolites of sphingomyelin, as well as calcium ion fluxes, have a profound role in cellular signaling in almost all cell types. In addition, metabolites of sphingomyelin often modulate calcium signaling, either directly or indirectly. This is an interesting aspect on how lipids may wield their physiological role, as calcium is probably one of the most versatile signaling molecules in the cell, and as modulation of calcium signaling has profound effects on cellular physiology. The aim of this review is to discuss the mechanisms by which metabolites of sphingomyelin, especially the sphingolipids sphingosine and sphingosine 1-phosphate (S1P), modulate calcium fluxes, and how this may affect cellular function. In addition, the pathological aspects of sphingolipid-evoked modulation of calcium fluxes will be discussed.

In vitro and in vivo characterization of modulation of the vacuolar cation channel TRPY1 from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses a transient receptor potential (TRP) channel homolog TRPY1 in its vacuolar membrane, considered to be an ancestral TRP channel. So far, studies have focused on the channel properties of TRPY1, but its regulation and physiologic role remained to be elucidated. Here, we investigated TRPY1 channel function in vitro and in vivo. Patch-clamp recording of TRPY1 in yeast vacuolar membranes showed that Ca2+ on the lumen side inhibited TRPY1-mediated channel activity, whereas luminal Zn2+ increased the currents. TRPY1 was activated in the presence of a reducing agent, 2-mercaptoethanol. The cysteine at position 624 was identified as the target for this activating action. This activation was independent of the presence of cytosolic Ca2+ . The amplitude of TRPY1-mediated current was reduced by addition of phosphatidylinositol 3-phosphate on the cytosolic side but not by phosphatidylinositol (PI) or phosphatidylinositol 3,5-phosphate. Measurement of the transient Ca2+ increase in response to hyper-osmotic shock in several yeast mutants defective in different steps of the PI phosphate biogenesis pathway supported this interpretation. Addition of a microtubule inhibitor strongly decreased the transient cytosolic Ca2+ increase upon hyper-osmotic shock. Taken together, the data indicate that the vacuolar TRPY1 Ca2+ channel mediates the perception of cytosolic signals that were induced by external changes in osmolarity, and participates in the modulation of cytosolic calcium signaling through Ca2+ release from the vacuole to maintain intracellular Ca2+ homeostasis in yeast.

Hepatitis B virus X protein modulates cytosolic Ca2+ signaling in primary human hepatocytes

Worldwide, approximately 240 million people are chronically infected with the hepatitis B virus (HBV); chronic HBV infection is associated with the development of life-threatening liver diseases. The HBV HBx protein alters hepatocyte physiology to promote HBV replication. We previously reported that HBx modulates calcium signaling to stimulate HBV replication in human hepatoblastoma HepG2 cells and primary rat hepatocytes. Whether HBx modulates calcium signaling in a primary human hepatocyte, the natural site of an HBV infection, has not been determined. Here, we report the effect of HBx on calcium signaling in primary human hepatocytes and show that HBx modulates calcium signaling via enhanced calcium entry through store-operated calcium channels and elevated mitochondrial calcium, similar to HBx effects in HepG2 cells and primary rat hepatocytes. In addition to demonstrating that HBV and HBx affect calcium signaling in human hepatocytes, these studies also show that HBV and HBx regulation of calcium signaling is identical in primary human and rat hepatocytes, further validating the use of cultured primary rat hepatocytes for HBV studies.

Taurine: A potential mediator for periodontal therapy.

Taurine or 2-aminoethanesulfonic has many fundamental biological roles such as conjugation of bile acids, antioxidation, osmoregulation, membrane stabilization, and modulation of calcium signaling. It is essential for cardiovascular function and development and function of the skeletal muscle, the retina, and the central nervous system. Functions of taurine include osmoregulation; membrane stabilization; modulation of calcium levels; and antioxidation, antiapoptotic, anti-inflammatory, and antilipid activities. Taurine was first discovered as a component of ox (Bos taurus, from which its name is derived) bile in 1827; it had taken over a century before insights into its physiological functions were made. The present review throws light on the multifactorial properties of taurine and its potential to be used in periodontal therapy.

MAP6 interacts with Tctex1 and Cav 2.2/N-type calcium channels to regulate calcium signalling in neurons.

MAP6 proteins were first described as microtubule‐stabilizing agents, whose properties were thought to be essential for neuronal development and maintenance of complex neuronal networks. However, deletion of all MAP6 isoforms in MAP6 KO mice does not lead to dramatic morphological aberrations of the brain but rather to alterations in multiple neurotransmissions and severe behavioural impairments. A search for protein partners of MAP6 proteins identified Tctex1 – a dynein light chain with multiple non‐microtubule‐related functions. The involvement of Tctex1 in calcium signalling led to investigate it in MAP6 KO neurons. In this study, we show that functional Cav2.2/N‐type calcium channels are deficient in MAP6 KO neurons, due to improper location. We also show that MAP6 proteins interact directly with both Tctex1 and the C‐terminus of Cav2.2/N‐type calcium channels. A balance of these two interactions seems to be crucial for MAP6 to modulate calcium signalling in neurons.

NOTCH1 is a mechanosensor in adult arteries

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell–cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.

FTY-720 induces apoptosis in neuroblastoma via multiple signaling pathways.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor. High-risk NB is difficult to treat due to the lack of response to current therapies and aggressive disease progression. Despite novel drugs, alternative treatments and multi-modal treatments, finding an effective treatment strategy for these patients continues to be a major challenge. The current study focuses on examining the effects of FTY-720 or fingolimod, a drug that is FDA-approved for refractory multiple sclerosis, in NB. The results showed that FTY-720 regulates multiple pathways that result in various effects on calcium signaling, ion channel activation and cell survival/death pathways. FTY-720 rapidly inhibits TRPM7 channel activity, and inhibited TRPM7 kinase activity, modulates calcium signaling, induces a loss of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore, and ultimately leads to cell death. Interestingly, the data also showed that low concentrations of FTY-720 sensitized drug-resistant NB cells to antineoplastic drugs. These results suggest that FTY-720 may be an attractive alternative for the treatment of NB.

Treatment of rotenone induced neurodegeneration by taurine and hesperidin

Taurine (2-aminoethane sulphonic acid) is a sulphonic acid which is derived from cysteine and is widely distributed in animal tissues. It is one of the most abundant amino acid in mammals and plays several crucial roles including modulation of calcium signaling, osmoregulation and membrane stabilization. Hesperidin occurs in the cells in crystalline, feather-like aggregates or sphaerocrytalline masses and it exhibits pharmacological and biological properties such as anti-inflammatory, anticarcinogenic, inhibit bone loss, lowering of lipid, hypoglycaemic and antioxidant activities. The current study was performed to evaluate the effect of taurine and hesperidin on neurodegeneration resulted from rotenone administration by a dose of 1.5 mg/kg b.wt three times per week for two months. Also we summarize recent findings emphasizing the role of catecholamines neurotransmitters, Tyrosine hydroxylase and oxidative stress in neurodegenerative disease model. These rats received taurine and hesperidin through gastric intubation daily for one month after rotenone administration. The results revealed that taurine and hesperidin treatment significantly ameliorated the decreased levels of the catecholamines neurotransmitters and Tyrosine hydroxylase which were decreased as after rotenone injection. Moreover, taurine and hesperidin treatment ameliorated lipid peroxidation and catalase levels.

Abstract 1346: Pan-ERBB inhibitor blocks tumor growth and metastasis

Abstract The Epidermal Growth Factor Receptor family of transmembrane tyrosine kinases (ErbB) is over-expressed, correlates with poor prognosis and negatively correlates with disease free survival in many cancer types. EGFR, HER2 and ErbB3 are known to be highly expressed and active, and have been targeted by both kinase inhibitors and monoclonal antibodies, both with disappointing results. Importantly, though, these therapeutics only target either a single receptor or the kinase domain alone. It is well known that these receptors function as heterodimers, and blocking of a single receptor will only promote the hetero-dimerization of the remaining family members. Furthermore, while the tyrosine kinase activity of these receptors is well known, less appreciated are the kinase-independent mechanisms by which they drive cancer progression. These include the modulation of calcium signaling and mitochondrial and nuclear translocation and activity as transcriptional co-factors. The highly conserved juxta-membrane domain (JD) of the ErbB receptors regulates these kinase-independent functions, as well as receptor homo- and hetero-dimerization. Upon ligand binding, the JD forms anti-parallel dimers between the receptors, resulting in receptor dimerization and intracellular trafficking. These protein-protein interactions result in cell growth, survival, migration and invasion. We have previously shown that peptides mimicking this JD can act in a dominant-negative fashion, promoting the formation of non-functional ErbB dimers (consisting of EGFR, HER2 and ErbB3) that induce rapid, ErbB-dependent cell death. Using Cell Penetrating Peptides synthesized in tandem with the JD, these peptides (called EJ1) rapidly cross the plasma membrane, bind the ErbB receptors, and induce cell death in cell lines and PDX lines grown in culture. Death results from a combination of apoptotic and necrotic mechanisms, due to inactivation of mitochondrial function and calcium signaling (as well as kinase inhibition). Although EJ1 peptides display rapid ErbB-dependent cell death in vitro, they are rapidly cleared in vivo, limiting their efficacy. To stabilize in vivo activity, the active peptide (which is an alpha-helix) was stabilized with hydrocarbon staples. Based on this, we hypothesized that SAH-EJ1 would be an effective targeted therapeutic for the treatment of ErbB-dependent cancer. We therefore tested SAH-EJ1 in 3 different ErbB-dependent models of cancer, including EGFRvIII-driven glioblastoma, a PDX model of basal-like breast cancer with EGFR and ErbB3 amplification, and a PDX model of lung cancer with EGFR kinase domain mutations driven by Erlotinib treatment. In each of these models, substantial inhibition of tumor growth was observed, as well as extended survival. Citation Format: Joyce A. Schroeder, Sabrina Maisel, Derrick Broka. Pan-ERBB inhibitor blocks tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1346. doi:10.1158/1538-7445.AM2017-1346

P2X7 receptor large pore signaling in avian Müller glial cells.

ATP is a pleiotropic molecule that promotes extra- and intracellular signaling to regulate numerous functions. This nucleotide activates purine and pyrimidine receptors at the plasma membrane, categorized as ionotropic P2X or G-protein-coupled receptor (GPCR) P2Y receptors. P2X are ligand-gated ion channel receptors, expressed in both retinal neurons and Müller cells leading to neuron-glia communication, calcium waves and neurovascular coupling. However, how P2X pore is formed upon ATP activation and how signaling pathways regulates the complex is still a matter of controversy. Here we studied the properties of the P2X7 receptor (P2X7R) using electrophysiology, single cell Ca2+ imaging, and dye uptake assay in purified avian Müller glia in culture. Our data show that ATP (or benzoyl-benzoyl ATP, BzATP) evoked large inward currents in patch-clamp studies while addition of P2X7R antagonist such as brilliant Blue G (BBG), abolished these currents. Ruthenium red (RU-2), a general transient receptor potential (TRP) inhibitor, reduced currents induced by ATP. Our data also point to the involvement of mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), Ca2+-calmodulin kinase II (CAMKII), microtubules or protein kinase C (PKC) modulating ATP-induced ionic current in Müller cells. We show that ATP induced Ca2+ influx, partially inhibited by P2X7R antagonists (oxidized ATP or BBG), and totally inhibited by blockers of other pores such as transient receptor potential (TRPs) or connexin hemichannel. Additionally, MAPK, PKC, PI3K or CAMKII inhibitors also are involved in the modulation of intracellular calcium signaling. Finally, ATP induced 80-90% of dye uptake in Muller glia cells, while oxidized ATP (oATP), BBG or A740003 inhibited this effect. We conclude that large conductance channel and other P2XRs are not involved in the ATP-induced dye uptake, but signaling pathways such as MAPK, PI3-K, microtubules or PKC are involved in pore formation.

Abstract 170: Glucose Oxidation by Pyruvate Dehydrogenase Ameliorates Cradiomyocytes Contractility in Response to Hypoxic Stress

Introduction: Pyruvate dehydrogenase (PDH) plays a key role in aerobic energy metabolism and occupies a central crossroad between glycolysis and the tricarboxylic acid cycle. Dichloroacetate (DCA), a PDH activator, has been revealed to increase glucose oxidation and reduce myocardial infarct size during ischemia and reperfusion. Hypothesis: Cardiac PDH activation plays a critical role in modulation of cardiomyocytes contractility and calcium signaling in response to hypoxia. Methods: Mechanical properties and intracellular Ca 2+ homeostasis were measured in isolated cardiomyocytes by IonOptix system. The stress signaling was evaluated using immunoblotting and immunoprecipitation analysis. Results: PDH activator DCA treatment significantly protects cardiomyocytes from hypoxia-induced contractile dysfunction as measured by maximal velocity of shortening (+dL/dt) and relengthening (–dL/dt), peak height and peak shortening (PS) amplitude, time-to-90% relengthening (TR90) in cardiomyocytes. However, DCA treatment did not show any protective effects on contractile functions of isolated cardiomyocytes from the cardiomyocyte specific PDH KO mouse hearts under hypoxic conditions. Intriguingly, the rise of intracellular Ca 2+ levels and intracellular ATP levels have no significant difference among wild type (WT) and PDH KO cardiomyocytes with and without DCA treatment. The results demonstrated that cardiomyocyte PDH KO does not affect cardiomyocyte contractility under normal physiological conditions, but significantly impaired the contractile functions of isolated cardiomyocytes under hypoxic conditions. Furthermore, the immunoblotting results showed that hypoxia triggered phosphorylation of AMP-activated protein kinase (AMPK) in isolated cardiomyocytes, but the AMPK phosphorylation was significantly impaired in PDH KO cardiomyocytes. DCA treatment clearly augmented ischemic AMPK phosphorylation in the isolated wild type (WT) but not in PDH KO cardiomyocytes. Conclusions: The glucose oxidation by pyruvate dehydrogenase (PDH) plays a critical role in cardiomyocyte contractility in response to hypoxic stress. PDH agonist DCA could be used for improve contractile function of cardiomyocytes under hypoxic insults.

Stereoselective β-mannosylation via anomeric O-alkylation: Formal synthesis of potent calcium signal modulator acremomannolipin A

Abstract Stereoselective β-mannosylation has been investigated via cesium carbonate-mediated anomeric O-alkylation of d -mannose-derived lactol with various electrophiles. It was found that electrophiles bearing trifluoromethanesulfonate (triflate) as the leaving group are most reactive. In addition, a highly efficient formal synthesis of potent calcium signal modulator acremomannolipin A has been achieved using this β-mannosylation method.

Annexin/S100A Protein Family Regulation through p14ARF-p53 Activation: A Role in Cell Survival and Predicting Treatment Outcomes in Breast Cancer.

The annexin family and S100A associated proteins are important regulators of diverse calcium-dependent cellular processes including cell division, growth regulation and apoptosis. Dysfunction of individual annexin and S100A proteins is associated with cancer progression, metastasis and cancer drug resistance. This manuscript describes the novel finding of differential regulation of the annexin and S100A family of proteins by activation of p53 in breast cancer cells. Additionally, the observed differential regulation is found to be beneficial to the survival of breast cancer cells and to influence treatment efficacy. We have used unbiased, quantitative proteomics to determine the proteomic changes occurring post p14ARF-p53 activation in estrogen receptor (ER) breast cancer cells. In this report we identified differential regulation of the annexin/S100A family, through unique peptide recognition at the N-terminal regions, demonstrating p14ARF-p53 is a central orchestrator of the annexin/S100A family of calcium regulators in favor of pro-survival functions in the breast cancer cell. This regulation was found to be cell-type specific. Retrospective human breast cancer studies have demonstrated that tumors with functional wild type p53 (p53wt) respond poorly to some chemotherapy agents compared to tumors with a non-functional p53. Given that modulation of calcium signaling has been demonstrated to change sensitivity of chemotherapeutic agents to apoptotic signals, in principle, we explored the paradigm of how p53 modulation of calcium regulators in ER+ breast cancer patients impacts and influences therapeutic outcomes.

ATF3 modulates calcium signaling in osteoclast differentiation and activity by associating with c-Fos and NFATc1 proteins

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, whether ATF3 is involved in osteoclast differentiation and activity has not been well-studied. In the present study, we examined the role of ATF3 in osteoclast differentiation and function. ATF3 expression was down-regulated during RANKL-induced osteoclast differentiation. Overexpression of ATF3 in bone marrow-derived monocyte/macrophage lineage cells (BMMs) promoted osteoclast differentiation and activity and strongly induced the expression of osteoclast genes encoding nuclear factor of activated T-cells c1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP) compared to that in the control group. In contrast, small interfering RNA-mediated knockdown of ATF3 prevented the formation of multinucleated osteoclasts and markedly abrogated the expression of osteoclast marker genes. Mechanistically, ATF3 synergistically enhanced c-Fos- or NFAT-mediated transcriptional activity of the NFATc1 or TRAP promoter, respectively. Furthermore, ATF3 physically interacted with c-Fos and NFATc1 and enhanced the binding affinity of c-Fos and NFATc1 to the promoters. Interestingly, ATF3 is involved in calcium signaling during osteoclastogenesis. Taken together, these results suggest that ATF3 is a new co-factor of c-Fos and NFATc1 to activate osteoclast differentiation and activity.

Unveiling (−)‐Englerin A as a Modulator of L‐Type Calcium Channels

AbstractThe voltage‐dependent L‐type Ca2+channel was identified as a macromolecular target for (−)‐englerin A. This finding was reached by using an unprecedented ligand‐based prediction platform and the natural product piperlongumine as a pharmacophore probe. (−)‐Englerin A features high substructure dissimilarity to known ligands for voltage‐dependent Ca2+ channels, selective binding affinity for the dihydropyridine site, and potent modulation of calcium signaling in muscle cells and vascular tissue. The observed activity was rationalized at the atomic level by molecular dynamics simulations. Experimental confirmation of this hitherto unknown macromolecular target expands the bioactivity space for this natural product and corroborates the effectiveness of chemocentric computational methods for prioritizing target‐based screens and identifying binding counterparts of complex natural products.