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Cancer cells that express the receptor protein RANK migrate to bone tissue where the RANK ligand, RANKL, is found. This contributes to bone metastases and offers a potential therapeutic approach to prevent cancer metastases. Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain 1 , 2 , 3 . Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile ‘soil’ 4 . In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs 5 . However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour 6 , other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-κB ligand) 7 , 8 triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis 9 , in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.

Systemic acidosis has detrimental effects on the skeleton, and local acidosis coincides with bone destruction in inflammatory and metastatic diseases. Acidification dramatically enhances osteoclastic resorption, although the underlying mechanism has remained elusive. We investigated the effect of acidosis on the osteoclastogenic transcription factor NFATc1, which upon dephosphorylation translocates from the cytoplasm to nuclei. Lowering extracellular pH dramatically increased accumulation of NFATc1 in nuclei of rat and rabbit osteoclasts to levels comparable with those induced by the proresorptive cytokine receptor activator of NF-κB ligand (RANKL). Activation of NFATc1 by RANKL was mediated by means of prolonged stimulation of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In contrast, NFATc1 activation by acidosis involved stimulation of calcineurin and suppression of NFATc1 inactivation. Acidosis, like RANKL, induced transient elevation of cytosolic free Ca2+concentration ([ Ca2+]i), which persisted in Ca2+-free media and was abolished by inhibition of phospholipase C or depletion of intracellular Ca2+stores. Real-time-PCR of osteoclast-like cells generated from RAW 264.7 cells revealed high levels of expression of ovarian cancer G protein-coupled receptor 1, which links extracellular acidification to elevation of [ Ca2+]i. In addition, the calcineurin inhibitor cyclosporin A suppressed the stimulatory effect of acidification on resorption, implicating NFAT in mediating the actions of acidosis on osteoclast activity. In summary, acidification and RANKL induce signals in osteoclasts that converge on the Ca2+/calcineurin/ NFAT pathway. Acidosis acts directly on osteoclasts to activate NFATc1 and stimulate resorption.

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca 2+ , which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca 2+ handling, Ca 2+ transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1 CKO ) and wild-type (WT) mice were determined. LPS increased intracellular Ca 2+ in WT but not in Rac1 CKO cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca 2+ /CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.

Chronic ginseng treatments have been purported to improve cardiac performance. However reports of acute administration of ginseng on cardiovascular function remain controversial and potential mechanisms are not clear. In this study, we examined the effects of acute North American ginseng (Panax quinquefolius) administration on rat cardiac contractile function by using electrocardiogram (ECG), non-invasive blood pressure (BP) measurement, and Langendorff isolated, spontaneously beating, perfused heart measurements (LP). Eight-week old male Sprague-Dawley rats (n = 8 per group) were gavaged with a single dose of water-soluble American ginseng at 300 mg/kg body weight. Heart rate (HR) and BP were measured prior to and at 1 and 24 h after gavaging (ECG and BP). Additional groups were used for each time point for Langendorff measurements. HR was significantly decreased (ECG: 1 h: 6 ± 0.2%, 24 h: 8 ± 0.3%; BP: 1 h: 8.8 ± 0.2%, 24 h: 13 ± 0.4% and LP: 1 h: 22 ± 0.4%, 24 h: 19 ± 0.4%) in rats treated with water-soluble ginseng compared with pre or control measures. An initial marked decrease in left ventricular developed pressure was observed in LP hearts but BP changes were not observed in BP group. A direct inhibitory effect of North American ginseng was observed on cardiac contractile function in LP rats and on fluorescence measurement of intracellular calcium transient in freshly isolated cardiac myocytes when exposed to ginseng (1 and 10 μg/ml). Collectively these data present evidence of depressed cardiac contractile function by acute administration of North American ginseng in rat. This acute reduction in cardiac contractile function appears to be intrinsic to the myocardium.

Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10 −4 N.m −1 were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.

Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload‐induced Ca2+ release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1f/f and cardiac‐specific Rac1 knockdown (Rac1ckd) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1f/f mice. Remarkably, I/R‐induced ventricular arrhythmia was significantly decreased in Rac1ckd compared to Rac1f/f mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R‐induced ventricular arrhythmia. Ca2+ imaging analysis showed that in response to a 6 mM external Ca2+ concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca2+ waves in Rac1f/f cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R‐induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1ckd mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.

We describe a simple fabrication technique – targeted towards non-specialists – that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities.

Martin and Mundy emphasize the crucial role played by osteoclasts in the metastasis of many tumour types to bone. Our intention was not to question the importance of osteoclasts in bone metastases, but rather to show that RANK ligand (RANKL) exerts additional action on cancer cells beyond its effects on osteoclasts. We reported that epithelial tumour cells can express RANK, a finding that is supported by earlier work; that RANKL has chemotactic activity; and that in vivo inhibition of RANKL by osteoprotegerin reduces tumour burden in bone when assessed in a previously published experimental system. [PUBLICATION ABSTRACT]

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum (ER/SR). However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase (nNOS) is located in the SR of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. nNOS deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione (GSNO) inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49 and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+-depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity, and inhibits SOCE in cardiomyocytes.

The $\\beta $-adrenergic agonist isoproterenol and analogs of adenosine 3$^{\\prime}$,5$^{\\prime}$-monophosphate (cAMP) induced a potassium current, M current, in freshly dissociated gastric smooth muscle cells. Muscarinic agonists suppress this current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. Thus, M current can be induced by an agent and regulated in antagonistic fashion by $\\beta $-adrenergic and muscarinic systems.