Calcium Signaling In Response Research Articles

Intercellular calcium waves integrate hormonal control of glucose output in the intact liver.

Key points Sympathetic outflow and circulating glucogenic hormones both regulate liver function by increasing cytosolic calcium, although how these calcium signals are integrated at the tissue level is currently unknown.We show that stimulation of hepatic nerve fibres or perfusing the liver with physiological concentrations of vasopressin only will evoke localized cytosolic calcium oscillations and modest increases in hepatic glucose production.The combination of these stimuli acted synergistically to convert localized and asynchronous calcium responses into co‐ordinated intercellular calcium waves that spread throughout the liver lobule and elicited a synergistic increase in hepatic glucose production.The results obtained in the present study demonstrate that subthreshold levels of one hormone can create an excitable medium across the liver lobule, which allows global propagation of calcium signals in response to local sympathetic innervation and integration of metabolic regulation by multiple hormones. This enables the liver lobules to respond as functional units to produce full‐strength metabolic output at physiological levels of hormone. Glucogenic hormones, including catecholamines and vasopressin, induce frequency‐modulated cytosolic Ca2+ oscillations in hepatocytes, and these propagate as intercellular Ca2+ waves via gap junctions in the intact liver. We investigated the role of co‐ordinated Ca2+ waves as a mechanism for integrating multiple endocrine and neuroendocrine inputs to control hepatic glucose production in perfused rat liver. Sympathetic nerve stimulation elicited localized Ca2+ increases that were restricted to hepatocytes in the periportal zone. During perfusion with subthreshold vasopressin, sympathetic stimulation converted asynchronous Ca2+ signals in a limited number of hepatocytes into co‐ordinated intercellular Ca2+ waves that propagated across entire lobules. A similar synergism was observed between physiological concentrations of glucagon and vasopressin, where glucagon also facilitated the recruitment of hepatocytes into a Ca2+ wave. Hepatic glucose production was significantly higher with intralobular Ca2+ waves. We propose that inositol 1,4,5‐trisphosphate (IP3)‐dependent Ca2+ signalling gives rise to an excitable medium across the functional syncytium of the hepatic lobule, co‐ordinating and amplifying the metabolic responses to multiple hormonal inputs.

Novel high molecular weight albumin-conjugated angiotensin II activates β-arrestin and G-protein pathways.

To study the ability of a novel bovine serum albumin-angiotensin II (BSA-Ang II) conjugate to effect responses of the AT1 angiotensin II receptor subtype mediated by the G-protein-coupled and the beta-arrestin pathways. Angiotensin II (Ang II) was conjugated with bovine serum albumin and compared with Ang II for competition binding to AT1 receptors, to stimulate aldosterone release from adrenocortical cells, to promote beta-arrestin binding to AT1 receptors, to promote calcium mobilization, and stimulate drinking of water and saline by rats. The BSA-Ang II conjugate was less potent competing for AT1R binding, but was equally efficacious at stimulating aldosterone release from H295R adrenocortical cells. Both BSA-Ang II and Ang II stimulated calcium mobilization and beta-arrestin binding to AT1 receptors. BSA-Ang II and Ang II stimulated water appetite equivalently but BSA-Ang II stimulated saline appetite more than Ang II. Both BSA-Ang II and Ang II were considerably more potent at causing calcium mobilization than β-arrestin binding. Addition of a high molecular weight molecule to Ang II reduced its AT1 receptor binding affinity, but did not significantly alter stimulation of aldosterone release or water consumption. The BSA-Ang II conjugate caused a greater saline appetite than Ang II suggesting that it may be a more efficacious agonist of this beta-arrestin-mediated response than Ang II. The higher potency calcium signaling response suggests that the G-protein-coupled responses predominate at physiological concentrations of Ang II, while the beta-arrestin response requires pathophysiological or pharmacological concentrations of Ang II to occur.

Expression of maize calcium-dependent protein kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II.

In plants, CALCIUM-DEPENDENT PROTEIN KINASES (CDPKs/CPKs) are involved in calcium signaling in response to endogenous and environmental stimuli. Here, we report that ZmCPK11, one of maize CDPKs, participates in salt stress response and tolerance. Salt stress induced expression and upregulated the activity of ZmCPK11 in maize roots and leaves. Activation of ZmCPK11 upon salt stress was also observed in roots and leaves of transgenic Arabidopsis plants expressing ZmCPK11. The transgenic plants showed a long-root phenotype under control conditions and a short-root phenotype under NaCl, abscisic acid (ABA) or jasmonic acid (JA) treatment. Analysis of ABA and JA content in roots indicated that ZmCPK11 can mediate root growth by regulating the levels of these phytohormones. Moreover, 4-week-old transgenic plants were more tolerant to salinity than the wild-type plants. Their leaves were less chlorotic and showed weaker symptoms of senescence accompanied by higher chlorophyll content and higher quantum efficiency of photosystem II. The expression of Na+ /K+ transporters (HKT1, SOS1 and NHX1) and transcription factors (CBF1, CBF2, CBF3, ZAT6 and ZAT10) with known links to salinity tolerance was upregulated in roots of the transgenic plants upon salt stress. Furthermore, the transgenic plants accumulated less Na+ in roots and leaves under salinity, and showed a higher K+ /Na+ ratio in leaves. These results show that the improved salt tolerance in ZmCPK11-transgenic plants could be due to an upregulation of genes involved in the maintenance of intracellular Na+ and K+ homeostasis and a protection of photosystem II against damage.

Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells.

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature β cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived β cells that resemble their endogenous counterparts.

Increases in Absolute Temperature Stimulate Free Calcium Concentration Elevations in the Chloroplast.

Plants need to sense increases in temperature to be able to adapt their physiology and development to survive; however, the mechanisms of heat perception are currently relatively poorly understood. Here we demonstrate that in response to elevated temperature, the free calcium concentration of the stroma of chloroplasts increases. This response is specific to the chloroplast, as no corresponding increase in calcium is seen in the cytosol. The chloroplast calcium response is dose dependent above a threshold. The magnitude of this calcium response is dependent upon absolute temperature, not the rate of heating. This response is dynamic: repeated stimulation leads to rapid attenuation of the response, which can be overcome by sensitization at a higher temperature. More long-term acclimation to different temperatures resets the basal sensitivity of the system, such that plants acclimated to lower temperatures are more sensitive than those acclimated to higher temperatures. The heat-induced chloroplast calcium response was partially dependent upon the calcium-sensing receptor CAS which has been shown previously to regulate other chloroplast calcium signaling responses. Taken together, our data demonstrate the ability of chloroplasts to sense absolute high temperature and produce commensurately quantitative stromal calcium response, the magnitude of which is a function of both current temperature and stress history.

Abnormal response of costal chondrocytes to acidosis in patients with chest wall deformity

Costal cartilage is much understudied compared to the load bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken or pigeon chest respectively. A lack of understanding of the ultrastructural and molecular biology properties of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. Due to the avascular nature of cartilage, chondrocytes metabolize glycolytically, producing an acidic environment. During physical activity hydrogen ions move within cartilage driven by compressive forces, thus at any one time, chondrocytes experience transient changes in pH. A variety of ion channels on chondrocytes plasma membrane equip them to function in the rapidly changing conditions they experience. In this paper we describe reduced expression of the ASIC2 gene encoding the acid sensing ion channel isoform 2 (previously referred to as ACCN1 or ACCN) in patients with chest wall deformities. We hypothesized that chondrocytes from these patients cannot respond normally to changes in pH that are an integral part of the biology of this tissue. Activation of ASICs indirectly creates a cascade ultimately dependent on intracellular calcium transients. The objective of this paper was to compare internal calcium signaling in response to external pH changes in costal chondrocytes from patients with chest wall deformities and healthy individuals. Although the molecular mechanism through which chondrocytes are regulated by acidosis remains unknown, we observed reduced amplitudes of calcium rise in patient chondrocytes exposed to low pH that become further impaired upon repeat exposure.

Plant Calcium Signaling in Response to Potassium Deficiency.

Potassium (K+) is an essential macronutrient of living cells and is the most abundant cation in the cytosol. K+ plays a role in several physiological processes that support plant growth and development. However, soil K+ availability is very low and variable, which leads to severe reductions in plant growth and yield. Various K+ shortage-activated signaling cascades exist. Among these, calcium signaling is the most important signaling system within plant cells. This review is focused on the possible roles of calcium signaling in plant responses to low-K+ stress. In plants, intracellular calcium levels are first altered in response to K+ deficiency, resulting in calcium signatures that exhibit temporal and spatial features. In addition, calcium channels located within the root epidermis and root hair zone can then be activated by hyperpolarization of plasma membrane (PM) in response to low-K+ stress. Afterward, calcium sensors, including calmodulin (CaM), CaM-like protein (CML), calcium-dependent protein kinase (CDPK), and calcineurin B-like protein (CBL), can act in the sensing of K+ deprivation. In particular, the important components regarding CBL/CBL-interacting protein kinase (CBL/CIPK) complexes-involved in plant responses to K+ deficiency are also discussed.

Impaired myogenic development, differentiation and function in hESC-derived SMA myoblasts and myotubes.

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells.

Assessing real-time signaling and agonist-induced CRHR1 internalization by optical methods.

The development of live-cell sensors for real-time measurement of signaling responses, with improved spatial and temporal resolution with respect to classical biochemical methods, has changed our understanding of cellular signaling. Examination of cAMP generation downstream activated GPCRs has shown that signaling responses can be short-lived (generated from the cell surface) or prolonged after receptor internalization. Class B secretin-like Corticotropin-releasing hormone receptor 1 (CRHR1) is a key player in stress pathophysiology. By monitoring real-time signaling in living cells, we uncovered cell context-dependent temporal characteristics of CRHR1-elicited cAMP responses and disclosed a specific link between cAMP generation and receptor signaling from internal compartments. We describe technical aspects and elaborate the protocols for cell line expression of Förster resonance energy transfer (FRET)-based biosensors to study the dynamics of cAMP and calcium signaling responses downstream activated CRHR1, live-cell imaging and analysis, and fluorescence flow cytometry to determine receptor levels at the cell surface.

Protease-activated receptor 1 is implicated in irritable bowel syndrome mediators-induced signaling to thoracic human sensory neurons.

Proteases and protease-activated receptors (PARs) are major mediators involved in irritable bowel syndrome (IBS). Our objectives were to decipher the expression and functionality (calcium signaling) of PARs in human dorsal root ganglia (DRG) neurons and to define mechanisms involved in human sensory neuron signaling by IBS patient mediators. Human thoracic DRG were obtained from the national disease resource interchange. Expression of PAR1, PAR2, and PAR4 was assessed by immunohistochemistry and quantitative reverse transcription PCR (RT-qPCR) in whole DRG or in primary cultures of isolated neurons. Calcium signaling in response to PAR agonist peptides (PAR-AP), their inactive peptides (PAR-IP), thrombin (10 U/mL), supernatants from colonic biopsies of patients with IBS, or healthy controls, with or without PAR1 or PAR4 antagonist were studied in cultured human DRG neurons. PAR1, PAR2, and PAR4 were all expressed in human DRG, respectively, in 20%, 40%, and 40% of the sensory neurons. PAR1-AP increased intracellular calcium concentration in a dose-dependent manner. This increase was inhibited by PAR1 antagonism. By contrast, PAR2-AP, PAR4-AP, and PAR-IP did not cause calcium mobilization. PAR1-AP-induced calcium flux was significantly reduced by preincubation with PAR4-AP, but not with PAR2-AP. Thrombin increased calcium flux, which was inhibited by a PAR1 antagonist and increased by a PAR4 antagonist. Supernatants from colonic biopsies of patients with IBS induced calcium flux in human sensory neurons compared with healthy controls, and this induction was reversed by a PAR1 antagonist. Taken together, our results highlight that PAR1 antagonism should be investigated as a new therapeutic target for IBS symptoms.

Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models.

SummaryIn vivo studies of human brain cellular function face challenging ethical and practical difficulties. Animal models are typically used but display distinct cellular differences. One specific example is astrocytes, recently recognized for contribution to neurological diseases and a link to the genetic risk factor apolipoprotein E (APOE). Current astrocytic in vitro models are questioned for lack of biological characterization. Here, we report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. We characterized NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs through transcriptomics, proteomics, glutamate uptake, inflammatory competence, calcium signaling response, and APOE secretion. Finally, we assess modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Our data show large diversity among astrocytic models and emphasize a cellular context when studying astrocyte biology.

Evaluation of initiating characteristics of osteoblastic calcium signaling responses to stretch by video rate time-course observation

Osteoblasts change their intracellular calcium ion concentration in response to mechanical stimuli. Although it has been reported that osteoblasts sense and respond to stretching of a substrate on which osteoblastic cells have adhered, the details of the dynamic characteristics of their calcium signaling response remain unclear. Motion artifacts such as loss of focus during stretch application make it difficult to conduct precise time-course observations of calcium signaling responses. Therefore, in this study, we observed intracellular calcium signaling responses to stretch in a single osteoblastic cell by video rate temporal resolution. Our originally developed cell-stretching microdevice enables in situ observation of a stretched cell without excessive motion artifacts such as focus drift. Residual minor effects of motion artifacts were corrected by the fluorescence ratiometric method with fluorescent calcium indicator Fluo 8H and fluorescent cytoplasm dye calcein red-orange. We succeeded to detect the intracellular calcium signaling response to stretch by video rate temporal resolution. The results revealed a time lag from stretch application to initiation of the intracellular calcium signaling response. We compared two time lags measured at two different cell areas: central and peripheral regions of the cell. The time lag in the central region of the cell was shorter than that in the peripheral region. This result suggests that the osteoblastic calcium signaling response to stretching stimuli initiates around the central region of the cell.

Lung tissue gene-expression signature for the ageing lung in COPD

IntroductionCOPD is a chronic, progressive, inflammatory disease of the lungs and the third leading cause of death worldwide. The current knowledge of the pathophysiology of COPD is limited and novel...

Fatiguing Contractions Induce Acute Redox Signaling in Mouse Muscle

Protein S-glutathionylation (P-SSG) is a reversible redox modification known to alter the activity of transcription factors, kinases, and contractile proteins as well as protect critical cysteine residues from irreversible oxidation. Exercise elicits an acute oxidative stress that is reported to result in beneficial adaptive responses, a process known as redox signaling. We hypothesized that redox signaling will occur through P-SSG of regulatory cysteines following fatiguing muscle contractions. To test this hypothesis, we performed acute fatiguing contractions using an in vivo stimulation protocol on anesthetized CB6F1 (BALB/cBy x C57BL/6) mice. Muscle force production was monitored by a force transducer throughout the 15 minutes of fatiguing stimulations. The right (stimulated) and left (unstimulated) gastrocnemius were collected 60 minutes after the last stimulation and processed for redox proteomics using resin-assisted thiol enrichment and selective reduction. Redox proteomics revealed over 1,500 P-SSG modifications that were significantly increased by fatiguing contractions. Mitochondrial proteins were among the most sensitive to glutathionylation. Ingenuity Pathway Analysis revealed mitochondrial dysfunction, calcium signaling, cytoskeleton signaling, and oxidative stress response in the top ten affected pathways. Critical cysteines on beta-actin, troponin I, ryanodine receptor 1, SERCA1, ATP synthase subunit alpha and NADH dehydrogenase flavoprotein 1 were among those significantly glutathionylated that have previously been reported to alter protein and muscle function. These modifications of redox sensitive proteins implicate rapid regulation of metabolism, excitation/contraction coupling, kinase activity, and contractile protein function, and protection from irreversible oxidation. Together, these data suggest a direct role of ROS in acute exercise signaling and map the redox sensitive proteome in mouse muscle. This study lays the groundwork for future investigation into the redox signaling of altered exercise adaptation associated with chronic conditions, such as sarcopenia, the age-related loss of muscle mass and function.

Cytosolic and Nucleosolic Calcium Signaling in Response to Osmotic and Salt Stresses Are Independent of Each Other in Roots of Arabidopsis Seedlings.

Calcium acts as a universal second messenger in both developmental processes and responses to environmental stresses. Previous research has shown that a number of stimuli can induce [Ca2+] increases in both the cytoplasm and nucleus in plants. However, the relationship between cytosolic and nucleosolic calcium signaling remains obscure. Here, we generated transgenic plants containing a fusion protein, comprising rat parvalbumin (PV) with either a nuclear export sequence (PV-NES) or a nuclear localization sequence (NLS-PV), to selectively buffer the cytosolic or nucleosolic calcium. Firstly, we found that the osmotic stress-induced cytosolic [Ca2+] increase (OICIcyt) and the salt stress-induced cytosolic [Ca2+] increase (SICIcyt) were impaired in the PV-NES lines compared with the Arabidopsis wildtype (WT). Similarly, the osmotic stress-induced nucleosolic [Ca2+] increase (OICInuc) and salt stress-induced nucleosolic [Ca2+] increase (SICInuc) were also disrupted in the NLS-PV lines. These results indicate that PV can effectively buffer the increase of [Ca2+] in response to various stimuli in Arabidopsis. However, the OICIcyt and SICIcyt in the NLS-PV plants were similar to those in the WT, and the OICInuc and SICInuc in the PV-NES plants were also same as those in the WT, suggesting that the cytosolic and nucleosolic calcium dynamics are mutually independent. Furthermore, we found that osmotic stress- and salt stress-inhibited root growth was reduced dramatically in the PV-NES and NLS-PV lines, while the osmotic stress-induced increase of the lateral root primordia was higher in the PV-NES plants than either the WT or NLS-PV plants. In addition, several stress-responsive genes, namely CML37, DREB2A, MYB2, RD29A, and RD29B, displayed diverse expression patterns in response to osmotic and salt stress in the PV-NES and NLS-PV lines when compared with the WT. Together, these results imply that the cytosolic and nucleosolic calcium signaling coexist to play the pivotal roles in the growth and development of plants and their responses to environment stresses.

Calcium signalling in medial intercalated cell dendrites and spines.

Dendritic and spine calcium imaging in combination with electrophysiology in acute slices revealed that in medial intercalated cells of the amygdala: Action potentials back-propagate into the dendritic tree, but due to the presence of voltage-dependent potassium channels, probably Kv4.2 channels, attenuate over distance. A mixed population of AMPA receptors with rectifying and linear I-V relations are present at individual spines of a single neuron. Decay kinetics and pharmacology suggest tri-heteromeric NMDA receptors at basolateral-intercalated cell synapses. NMDA receptors are the main contributors to spine calcium entry in response to synaptic stimulation. Calcium signals in response to low- and high-frequency stimulation, and in combination with spontaneous action potentials are locally restricted to the vicinity of active spines. Together, these data show that calcium signalling in these GABAergic neurons is tightly controlled and acts as a local signal. The amygdala plays a central role in fear conditioning and extinction. The medial intercalated (mITC) neurons are GABAergic cell clusters interspaced between the basolateral (BLA) and central amygdala (CeA). These neurons are thought to play a key role in fear and extinction, controlling the output of the CeA by feed-forward inhibition. BLA to mITC cell inputs are thought to undergo synaptic plasticity, a mechanism underlying learning, which is mediated by NMDA receptor-dependent mechanisms that require changes in cytosolic calcium. Here, we studied the electrical and calcium signalling properties of mITC neurons in GAD67-eGFP mice using whole-cell patch clamp recordings and two-photon calcium imaging. We show that action potentials back-propagate (bAP) into dendrites, and evoke calcium transients in both the shaft and the dendritic spine. However, bAP-mediated calcium rises in the dendrites attenuate with distance due to shunting by voltage-gated potassium channels. Glutamatergic inputs make dual component synapses on spines. At these synapses, postsynaptic AMPA receptors can have linear or rectifying I-V relationships, indicating that some synapses express GluA2-lacking AMPA receptors. Synaptic NMDA receptors had intermediate decay kinetics, and were only partly blocked by GuN2B selective blockers, indicating these receptors are GluN1/GluN2A/GluN2B trimers. Low- or high-frequency synaptic stimulation raised spine calcium, mediated by calcium influx via NMDA receptors, was locally restricted and did not invade neighbouring spines. Our results show that in mITC neurons, postsynaptic calcium is tightly controlled, and acts as a local signal.

Abstract 2998: Elotuzumab can costimulate NK cell responses by engaging SLAMF7

Abstract Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, CD319) protein, which is highly and uniformly expressed on multiple myeloma cells. Improved survival has been observed in multiple myeloma patients treated with Elo in combination with immunomodulatory drugs such as dexamethasone and lenalidomide. Previous work showed that the main mechanism of Elo action in vitro is CD16-mediated antibody dependent cellular cytotoxicity (ADCC) via Fc interaction with CD16 (FcγRIIIa) receptor on NK cells. However, SLAMF7 is also expressed on NK cells where it transmits an activating signal. We hypothesized that Elo can directly activate NK cells via SLAMF7 ligation. Utilizing non-fucosylated Elo that has higher affinity to CD16 and Elo mutant that has an Fc region mutation that abrogates binding to CD16, we confirmed that Elo promotes NK cell activation and degranulation in a CD16-dependent manner. Specifically, non-fucosylated Elo had higher potency, whereas Elo mutant did not stimulate degranulation or CD69 expression. To test for co-stimulatory effects of Elo and Elo mutant we measured calcium signaling responses triggered by those antibodies in combination with antibodies to engage the activating receptors NKp46 and NKG2D. Elo or Elo mut alone had no effect on calcium signaling, but when added in combination with multimeric engagement of NKp46 and NKG2D both antibodies co-stimulated calcium signaling responses. We conclude that Elo stimulates NK cells via interaction with CD16, but direct engagement with SLAMF7 can also transduce co-activating signals to potentiate calcium signals generated through NKp46 and NKG2D in a CD16-independent manner. Our results provide evidence that direct engagement with SLAMF7 by Elo has the potential to reduce the threshold of NK cell activation triggered through other activating receptors. Citation Format: Tatiana Pazina, Ashley Mentlik James, Alexander W. MacFarlane, Natalie A. Bezman, Robert F. Graziano, Michael D. Robbins, Adam D. Cohen, Kerry S. Campbell. Elotuzumab can costimulate NK cell responses by engaging SLAMF7 [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 2998. doi:10.1158/1538-7445.AM2017-2998

Fibroblast-like synoviocyte mechanosensitivity to fluid shear is modulated by interleukin-1α

Fibroblast-like synoviocytes (FLS) reside in the synovial membrane of diarthrodial joints and are exposed to a dynamic fluid environment that presents both physical and chemical stimuli. The ability of FLS to sense and respond to these stimuli plays a key role in their normal function, and is implicated in the alterations to function that occur in osteoarthritis (OA). The present work characterizes the response of FLS to fluid flow-induced shear stress via real-time calcium imaging, and tests the hypothesis that this response is modulated by interleukin-1α (IL-1α), a cytokine elevated in OA. FLS demonstrated a robust calcium signaling response to fluid shear that was dose dependent upon stress level and required both external and internal calcium sources. Preconditioning with 10ng/mL IL-1α for 24h heightened this shear stress response by significantly increasing the percent of responding cells and peak magnitude, while significantly decreasing the time for a peak to occur. Intercellular communication via gap junctions was found to account for a portion of the FLS population response in normal conditions, and was significantly increased by IL-1α preconditioning. IL-1α was also found to significantly increase average length and incidence of the primary cilium, an organelle commonly implicated in shear mechanosensing. These findings suggest that the elevated levels of IL-1α found in the OA environment heighten FLS sensitivity to fluid shear by altering both intercellular communication and individual cell sensitivity, which could affect downstream functions and contribute to progression of the disease state.

The anti-SLAMF7 antibody elotuzumab mediates NK cell activation through both CD16-dependent and –independent mechanisms

ABSTRACTElotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab')2 or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective “trans”-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

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.