Calcium Release In Response Research Articles

Coagulation disorders in myocardial infarction with nonobstructive coronary arteries

Aim. To investigate the state of the platelet and plasma components of hemostasis in patients with myocardial infarction with non-obstructive coronary arteries (MINOCA).Material and methods. The study included 42 patients with non-ST-segment elevation myocardial infarction (NSTEMI): MINOCA (n=24) and MI-CAD (n=18). Platelet aggregation ability in response to activation was evaluated using Solar AP2110 and LASCA aggregometers. Platelet functional activity and calcium signaling were assessed using flow cytometry methods. The plasma component of hemostasis, in addition to routine coagulation tests was evaluated using the global coagulation test "Thrombodynamics". The control groups for tests consisted of healthy volunteers.Results. When analyzing the ability of platelets to form aggregates by the aggregometry tests, it was found that platelets in the MINOCA group formed aggregates significantly worse upon ADP stimulation at various concentrations compared to the MI-CAD group. However, when platelets were stimulated with collagen, the opposite effect was observed: in the MI-CAD group, there was a noticeable decrease in aggregate formation in terms of light scattering amplitude compared to the MINOCA group. Flow cytometry using the functional platelet activity test protocol revealed that both groups showed a significantly increased platelet size after activation, reduced platelet granularity) both at rest and upon activation, significantly decreased number of procoagulant phosphatidylserine-positive platelets, and reduced dense granule release upon activation compared to healthy volunteers. The calcium signaling test showed a weakened calcium release in response to ADP in the MINOCA group compared to the MI-CAD group. In the study of the plasma component, no significant differences between the groups or deviations were found according to both routine tests and the "Thrombodynamics" test.Conclusion. Platelet activity did not differ significantly between the MINOCA and MI-CAD groups; however, in the MINOCA group, platelet activity was lower in some tests compared to the MI-CAD group. In the study of the plasma hemostasis component, normocoagulation was recorded in both groups.

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

Genomic, Transcriptomic, and Proteomic Depiction of Induced Pluripotent Stem Cells-Derived Smooth Muscle Cells As Emerging Cellular Models for Arterial Diseases.

Vascular smooth muscle cells (SMCs) plasticity is a central mechanism in cardiovascular health and disease. We aimed at providing cellular phenotyping, epigenomic and proteomic depiction of SMCs derived from induced pluripotent stem cells and evaluating their potential as cellular models in the context of complex diseases. Human induced pluripotent stem cell lines were differentiated using RepSox (R-SMCs) or PDGF-BB (platelet-derived growth factor-BB) and TGF-β (transforming growth factor beta; TP-SMCs), during a 24-day long protocol. RNA-Seq and assay for transposase accessible chromatin-Seq were performed at 6 time points of differentiation, and mass spectrometry was used to quantify proteins. Both induced pluripotent stem cell differentiation protocols generated SMCs with positive expression of SMC markers. TP-SMCs exhibited greater proliferation capacity, migration and lower calcium release in response to contractile stimuli, compared with R-SMCs. Genes involved in the contractile function of arteries were highly expressed in R-SMCs compared with TP-SMCs or primary SMCs. R-SMCs and coronary artery transcriptomic profiles were highly similar, characterized by high expression of genes involved in blood pressure regulation and coronary artery disease. We identified FOXF1 and HAND1 as key drivers of RepSox specific program. Extracellular matrix content contained more proteins involved in wound repair in TP-SMCs and higher secretion of basal membrane constituents in R-SMCs. Open chromatin regions of R-SMCs and TP-SMCs were significantly enriched for variants associated with blood pressure and coronary artery disease. Both induced pluripotent stem cell-derived SMCs models present complementary cellular phenotypes of high relevance to SMC plasticity. These cellular models present high potential to study functional regulation at genetic risk loci of main arterial diseases.

Epigenetic and phenotypic characterization of iPSCs-derived smooth muscle cells: Towards a cellular model for complex arterial diseases

Smooth muscle cells (SMCs) capacity to switch between proliferative (synthetic) and quiescent (contractile) phenotypes is a widely studied mechanism in cardiovascular disease. Primary SMCs tend to lose many physiological features in culture, which makes the study of their contractile function challenging. Recently, an optimized protocol of induced pluripotent stem cells (iPSCs) differentiation into contractile SMCs was described. We aimed at obtaining a deep phenotyping of SMCs derived from iPSCs and evaluating these cellular models in the context of complex cardiovascular diseases. We differentiated 3 human iPSCs lines towards SMC phenotypes using two alternative 24-day protocols, involving either RepSox (R-SMCs) or TGF-β/PDGF-BB (TP-SMCs) during late differentiation. We analyzed gene expression and open chromatin profiles at 6 time points of differentiation and compared them to primary human SMCs and artery tissue. We used CRISPR-Cas9 system to induce mutations at LRP1 cardiovascular disease locus. Both differentiation protocols provided SMCs with abundant expression of typical SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile signals compared to R-SMCs. RNA-Seq results showed that most genes involved in the contractile function of arteries were highly expressed in R-SMCs, while TP-SMCs were more similar to cultured primary human SMCs. Open chromatin regions of R-SMCs were highly enriched for variants associated with vascular diseases such as hypertension and intracranial aneurysm, whereas TP-SMCs were more enriched for variants associated to peripheral artery disease and aortic aneurysm. Deletion of a cardiovascular disease associated enhancer region led to a dysregulation of LRP1 expression in TP-SMCs, but not in R-SMCs. LRP1 inactivation in TP-SMCs led to a major decrease in SMC proliferation, migration, and deregulation of multiple genes involved in SMC function and extracellular matrix maintenance. Differentiation of SMCs from iPSCs using two complementary protocols provides cellular models suitable for the study of a variety of vascular diseases. We confirmed LRP1 as a regulatory target gene of a common variant associated to multiple cardiovascular diseases and identified multiple genes involved in SMC function downstream of LRP1.

HIGHLIGHTS ACROSS MYOLOGY: O.13 Altered calcium handling in a zebrafish model of SELENON congenital muscular dystrophy

SELENON congenital muscular dystrophy (previously designated SEPN1 congenital muscular dystrophy) causes muscle weakness, scoliosis, and respiratory insufficiency in infants and children. Targeted treatments for this devastating disease are currently lacking. SELENON encodes the protein Selenoprotein N, a member of the selenoprotein family. Selenoprotein N is expressed at high levels in developing and immature tissues and adopts a ubiquitous, low-level expression pattern in mature tissues. This raises questions about how loss of Selenoprotein N leads to muscle-specific disease. Overlap in clinical and histopathologic characteristics between patients with SELENON congenital muscular dystrophy and certain types of RYR1 myopathy raise the possibility of interactions between Selenoprotein N and the RYR1 calcium channel. This possibility is supported by altered calcium release in SELENON-deficient myotubes in vitro but has not been tested in vivo. Here, calcium release was measured from Selenoprotein N-deficient zebrafish muscle fibers in vivo. Selenoprotein N-deficient zebrafish show decreased amplitude but increased frequency of calcium spikes during myogenesis with subsequent abnormal myofibril alignment in slow-twitch muscle fibers. Further, mature muscle fibers of Selenoprotein N-deficient zebrafish show decreased amplitude and duration of calcium release in response to depolarization compared to wildtype and control muscle fibers. The characteristics of calcium release in Selenoprotein N-deficient zebrafish muscle is suggestive of a leaky RYR1 calcium channel, as is typically seen in the setting of oxidative stress. These data support a role for Selenoprotein N in modulating RYR1 function in vivo, both during muscle development and during excitation-contraction coupling in mature muscle fibers.

Pharmacological Characteristics of Porcine Orexin 2 Receptor and Mutants.

Orexin receptors (OXRs) play a critical regulatory role in central control of food intake, maintenance of sleeping states, energy metabolism, and neuroendocrine homeostasis. However, most previous studies have focused on the sleep-promoting functions of OXRs in human beings, while their potential value in enhancing food intake for livestock breeding has not been fully exploited. In this study, we successfully cloned porcine orexin 2 receptor (pOX2R) complementary DNA and constructed four pOX2R mutants (P10S, P11T, V308I, and T401I) by site-directed mutagenesis, and their functional expressions were further confirmed through Western blotting analysis. Pharmacological characteristics of pOX2R and their mutants were further investigated. These results showed that the P10S, P11T, and T401I mutants had decreased cAMP signaling with orexin A, whereas only the P11T mutant decreased under the stimulation of orexin B. Besides, only P10S displayed a decreased calcium release in response to both orexin ligands. Importantly, these mutants exhibited decreased phosphorylation levels of ERK1/2, p38, and CREB to some degree compared with wild-type pOX2R. Collectively, these findings highlight the critical role of these mutations in pOX2R signaling and expand our understanding of molecular and pharmacological characterization of pOX2R.

D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson's disease.

Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.

A Matricryptic Conformation of the Integrin-Binding Domain of Fibronectin Regulates Platelet-Derived Growth Factor-Induced Intracellular Calcium Release.

Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5β1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5β1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin.

Assembly of fibronectin fibrils selectively attenuates platelet-derived growth factor–induced intracellular calcium release in fibroblasts

Cellular responses to platelet-derived growth factor (PDGF) are altered in a variety of pathological conditions, including cancers, fibroses, and vascular diseases, making PDGF-induced signaling pathways important therapeutic targets. The limited success of therapies designed to impact PDGF pathways may be overcome with a clearer understanding of how cells integrate signals from PDGF and the extracellular matrix (ECM). Here, we assessed the effects of fibronectin matrix assembly on the responsiveness of mesenchymal cells to PDGF. Our results indicate that fibroblast-mediated assembly of fibronectin fibrils attenuates intracellular calcium release in response to PDGF. The dose-dependent inhibition of PDGF-induced intracellular calcium release was specific to the ECM form of fibronectin. Further, a recombinant protein engineered to mimic ECM fibronectin similarly attenuated intracellular calcium release in response to PDGF. Of note, fibronectin attenuated the PDGF-calcium signaling axis at the level of phosphoinositide 3-kinase (PI3K) activation. Interestingly, ECM fibronectin did not alter other intracellular signals activated by PDGF, including activation of PDGF receptor β, AKT Ser/Thr kinase, phospholipase Cγ1, and extracellular signal-regulated kinase 1/2 (ERK1/2). Rather, fibronectin inhibited activation of the p55 regulatory subunit of PI3K in response to a variety of stimuli, indicating that ECM fibronectin selectively attenuates the intracellular calcium release cascade while leaving intact other PDGF signaling pathways. Selective regulation of calcium signaling by ECM fibronectin via the p55 regulatory subunit of PI3K represents a mechanism by which cells tune their response to PDGF and may therefore serve as a target to selectively regulate one branch of PDGF signaling.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis

Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2−/− mice. In Adgrl2+/− mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.

Epithelial Cells Induce a Cyclo-Oxygenase-1-Dependent Endogenous Reduction in Airway Smooth Muscle Contractile Phenotype.

Airway smooth muscle cells (ASMCs) are phenotypically regulated to exist in either a proliferative or a contractile state. However, the influence of other airway structural cell types on ASMC phenotype is largely unknown. Although epithelial cells are known to drive ASM proliferation, their effects on the contractile phenotype are uncertain. In the current study, we tested the hypothesis that epithelial cells reduce the contractile phenotype of ASMCs. To do so, we measured force production by traction microscopy, gene and protein expression, as well as calcium release by Fura-2 ratiometric imaging. ASMCs incubated with epithelial-derived medium produced less force after histamine stimulation. We observed reduced expression of myocardin, α-smooth muscle actin, and calponin within ASMCs after coculture with epithelial cells. Peak calcium release in response to histamine was diminished, and depended on the synthesis of cyclo-oxygenase-1 products by ASM and on prostaglandin E receptors 2 and 4. Together, these in vitro results demonstrate that epithelial cells have the capacity to coordinately reduce ASM contraction by functional antagonism and by reduction of the expression of certain contractile proteins.

Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47 phox and p40 phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

Abstract 388: Functional Role of Inositol 1,4,5-Trisphosphate Receptors in Cardiac Hypertrophy

Calcium is a second messenger that is essential for the control of a variety of cellular functions. In the heart, calcium plays an integral role in many cellular processes including energy metabolism, contraction, gene expression, and cell death. The inositol 1,4,5-trisphosphate receptor (IP 3 R) is a calcium channel that is ubiquitously expressed throughout the body. There are three IP 3 R isoforms encoded by separate genes. In the heart, the IP 3 R-2 isoform is reported to being most predominant with regards to both expression levels and functional significance. Several groups have shown that perinuclear IP 3 R-2 may play a key role in the progression of hypertrophy by mediating nuclear calcium release in response to endothelin-1 (ET-1) and angiotensin II. However, the functional roles of IP 3 R-1 and IP 3 R-3 in the heart are essentially unexplored despite measureable expression levels. We hypothesize that all three IP 3 R isoforms can contribute to cytosolic calcium transients and cardiac stress induced by ET-1. As our model we used dissociated neonatal and adult rat ventricular cardiomyocytes. We found readily detectable levels of expression of all three IP 3 Rs in both neonatal and adult rat ventricular cardiomyocytes. We found that in addition to the perinuclear area, all three IP 3 R isoforms were expressed throughout the cardiomyocyte including the sarcoplasmic reticulum. Using isoform specific siRNA, we found that knocking down individual IP 3 Rs did not alter ET-1 induced calcium responses in neonatal cardiomyocytes. However, knocking down all three isoforms abolished the effects of ET-1 on calcium homeostasis. We next tested whether IP 3 Rs mediate nuclear-specific calcium elevations. Using a genetically encoded calcium indicator targeted to the nucleus we were able to specifically discriminate nuclear versus cytosolic calcium transients after ET-1 treatment. Contrary to previous reports, we found no evidence that IP 3 R-mediated calcium release is confined to the nucleus. Our findings that IP 3 R isoforms are functionally redundant in the heart have significant implications for animal models of hypertrophy and human disease.

Abstract C60: Snail transcription factor contributes to bone metastasis in prostate and breast cancer cells

Abstract Prostate cancer that is hormone refractory and has metastasized preferentially to bone is the main cause of prostate cancer death, especially in African American men. Receptor activator of nuclear factor kappa B ligand (RANKL) and its receptor (RANK) contributes to bone metastatic lesions and bisphosphonates such as Zometa and Fosamax have been used as antagonists of RANKL for the treatment of breast and prostate cancer metastasis. African American men have the highest bone mineral density compared to any other race, and the role this may play in prostate cancer metastasis to bone is not clear. A better understanding of bone metastasis may lead to alternative treatment options for metastatic prostate cancer. Snail transcription factor is important early in development and in cancer cells and promotes cancer cell migration and progression by inducing epithelial mesenchymal transition (EMT). We have observed increased expression of Snail in prostate cancer bone metastatic human patient samples. We hypothesized that Snail can mediate EMT-mediated prostate cancer migration towards bone of high bone mineral density and mediate the vicious cycle of tumor-tumor microenvironment reciprocal interactions through calcium and RANKL signaling. We generated an EMT model for prostate and breast cancer utilizing the ARCaP human prostate and MCF-7 breast cancer cells overexpressing Snail and identified increased RANKL expression that was associated with increased osteoclastogenesis both in vitro and in vivo, as well as decreased bone volume and density. We utilized pre-molded bone discs which are allograft bone/polyurethane (PUR) composite bone void fillers with tunable properties that have advantage over existing bone implant models in that it contains bigger pore sizes that support rapid cellular infiltration and remodeling. We treated the bone discs with hydrochloric acid which decreased the bone density to a ratio of 1:1.18 for low (HCL-treated): high (untreated) bone density, which is quite close to the 1: 1.2 ratio seen in Caucasian vs African American men. Incubation of these bone discs with prostate or breast cancer cells overexpressing Snail led to increased calcium release from bone of high density as compared to low density. We are currently testing whether this increased calcium release in response to Snail may promote paracrine cell proliferation. Since Snail is not required by adult cells except during injury, targeting Snail that is mainly expressed by cancer cells may antagonize metastatic lesions in bone without affecting normal bone in other areas of the body. Citation Format: Basil A. Smith, Veronica Henderson, Christopher Coke, Jerald Dumas, Cimona Hinton, Manu Platt, Leland K. Chung, Majd Zayzafoon, Valerie A. Odero-Marah. Snail transcription factor contributes to bone metastasis in prostate and breast cancer cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C60. doi:10.1158/1538-7755.DISP13-C60

Calcium Release in Response to Local Nanomechancial Shear Stress Delivered by an AFM Tip

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Deficiency of G3BP1, the stress granules assembly factor, results in abnormal synaptic plasticity and calcium homeostasis in neurons

Ras-GAP SH3-domain-binding protein, G3BP, is an important component in the assembly of stress granules (SGs), which are cytoplasmic aggregates assembled following translational stress. To assess the physiological function of G3BP, we generated viable G3bp1-knockout (KO) mice, which demonstrated behavioral defects linked to the CNS-associated with ataxia phenotype. Immunohistochemistry pinpointed high expression of G3BP in the cytoplasm of hippocampal neurons and Purkinje cells of the cerebellum of wild-type mice. Also, electrophysiological measurements revealed that the absence of G3BP1 leads to an enhancement of short-term potentiation (STP) and long-term depression in the CA1 area of G3bp1 KO mice compared with wild-type mice. Consistently, G3BP1 deficiency in neurons leads to an increase in intracellular calcium and calcium release in response to (S)-3,5-Dihydroxyphenylglycine, a selective agonist of group I metabotropic glutamate receptors. These results show, for the first time, a requirement for G3BP1 in the control of neuronal plasticity and calcium homeostasis and further establish a direct link between SG formation and neurodegenerative diseases.

Characterizing Interactions between γ-Synuclein and PLCβ2 in Breast Cancer Cells

γ-Synuclein is a small intrinsically disordered protein of unknown function that is usually found in the peripheral nervous system. Phospholipase β2 (PLCβ2) is an important component of the G-protein signaling pathway and its activation leads to calcium release in cells. These two proteins are both overexpressed in late stage breast cancer but not in early stage (Stage I and II) or in healthy breast tissues. We have previously shown that γ-synuclein binds strongly to PLCβ2 both in-vivo and in-vitro, as demonstrated by immunoprecipitation, immunofluorescence and binding assays. Here, we show that knocking down the expression of γ-synuclein using siRNA, results in a concomitant reduction in the expression of PLCβ2. Here, we also find that reducing the level of γ-synuclein in MDA MB 231 cells (which mimics stage 4 breast cancer) causes significant changes in cell morphology that indicate a reversion to healthy breast cell phenotype. Additionally, such reduction of γ-synuclein in MDA MB 231 cells results in a stronger and faster calcium release in response to G protein stimulation, corroborating our previous work showing that the binding of γ-synuclein to PLCβ2 reduces its activity. We are currently investigating whether γ-synuclein promotes cell proliferation, migration and invasion through its association with PLCβ2. A peptide inhibitor of the γ-synuclein – PLCβ2 complex is presently being tested.This work was supported by NIH GM053132

Eif4a3 is required for accurate splicing of the Xenopus laevis ryanodine receptor pre-mRNA

The Exon Junction Complex (EJC) plays a critical role in multiple posttranscriptional events, including RNA subcellular localization, nonsense-mediated decay (NMD), and translation. We previously reported that knockdown of the EJC core component Eukaryotic initiation factor 4a3 (Eif4a3) results in full-body paralysis of embryos of the frog, Xenopus laevis. Here, we explore the cellular and molecular mechanisms underlying this phenotype. We find that cultured muscle cells derived from Eif4a3 morphants do not contract, and fail to undergo calcium-dependent calcium release in response to electrical stimulation or treatment with caffeine. We show that ryr (ryanodine receptor) transcripts are incorrectly spliced in Eif4a3 morphants, and demonstrate that inhibition of Xenopus Ryr function similarly results in embryonic paralysis. These results suggest that the EJC mediates muscle cell function via regulation of pre-mRNA splicing during early vertebrate embryogenesis.

OCRL1Modulates Cilia Length in Renal Epithelial Cells

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.