ORAI-mediated calcium entry: Mechanism and roles, diseases and pharmacology

ORAI1 is a protein located on the plasma membrane that acts as a calcium channel. Calcium enters via ORAI1 as a mechanism to refill the sarcoplasmic/endoplasmic reticulum calcium stores, the depletion of which can be detected by the sensor protein STIM1. Isoforms of these proteins ORAI2, ORAI3 and STIM2 also have roles in cellular calcium homeostasis but are less well characterized. This pathway of filling the calcium stores is termed store-operated calcium entry and while the pathway itself was proposed in 1986, the identity of the key molecular components was only discovered in 2005 and 2006. The characterization of the ORAI and STIM proteins has provided clearer information on some calcium-regulated pathways that are important in processes from gene transcription to immune cell function. Recent studies have also suggested the importance of the components of ORAI-mediated calcium entry in some diseases or processes significant in disease including the migration of breast cancer cells and thrombus formation. This review will provide a brief overview of ORAI-mediated calcium entry, its role in physiological and pathophysiological processes, as well as current and potential pharmacological modulators of the components of this important cellular calcium entry pathway. (C) 2010 Elsevier Inc. All rights reserved

UV-Induced DNA Damage Promotes Resistance to the Biotrophic Pathogen Hyaloperonospora parasitica in Arabidopsis1[C][OA]

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response

Expanding the genetic basis of copy number variation in familial breast cancer

Introduction: Familial breast cancer (fBC) is generally associated with an early age of diagnosis and a higher frequency of disease among family members. Over the past two decades a number of genes have been identified that are unequivocally associated with breast cancer (BC) risk but there remain a significant proportion of families that cannot be accounted for by these genes. Copy number variants (CNVs) are a form of genetic variation yet to be fully explored for their contribution to fBC. CNVs exert their effects by either being associated with whole or partial gene deletions or duplications and by interrupting epigenetic patterning thereby contributing to disease development. CNV analysis can also be used to identify new genes and loci which may be associated with disease risk. Methods: The Affymetrix Cytogenetic Whole Genome 2.7 M (Cyto2.7 M) arrays were used to detect regions of genomic re-arrangement in a cohort of 129 fBC BRCA1/BRCA2 mutation negative patients with a young age of diagnosis (<50 years) compared to 40 unaffected healthy controls (>55 years of age). Results: CNV analysis revealed the presence of 275 unique rearrangements that were not present in the control population suggestive of their involvement in BC risk. Several CNVs were found that have been previously reported as BC susceptibility genes. This included CNVs in RPA3, NBN (NBS1), MRE11A and CYP19A1 in five unrelated fBC patients suggesting that these genes are involved in BC initiation and/or progression. Of special interest was the identification of WWOX and FHIT rearrangements in three unrelated fBC patients. Conclusions: This study has identified a number of CNVs that potentially contribute to BC initiation and/or progression. The identification of CNVs that are associated with known tumour suppressor genes is of special interest that warrants further larger studies to understand their precise role in fBC

Copy Number Variation in Hereditary Non-Polyposis Colorectal Cancer

Hereditary non-polyposis colorectal cancer (HNPCC) is the commonest form of inherited colorectal cancer (CRC) predisposition and by definition describes families which conform to the Amsterdam Criteria or reiterations thereof. In ~50% of patients adhering to the Amsterdam criteria germline variants are identified in one of four DNA Mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Loss of function of any one of these genes results in a failure to repair DNA errors occurring during replication which can be most easily observed as DNA microsatellite instability (MSI)—a hallmark feature of this disease. The remaining 50% of patients without a genetic diagnosis of disease may harbour more cryptic changes within or adjacent to MLH1, MSH2, MSH6 or PMS2 or elsewhere in the genome. We used a high density cytogenetic array to screen for deletions or duplications in a series of patients, all of whom adhered to the Amsterdam/Bethesda criteria, to determine if genomic re-arrangements could account for a proportion of patients that had been shown not to harbour causative mutations as assessed by standard diagnostic techniques. The study has revealed some associations between copy number variants (CNVs) and HNPCC mutation negative cases and further highlights difficulties associated with CNV analysis

STaRRRT: a table of short tandem repeats in regulatory regions of the human genome

Background: Tandem repeats (TRs) are unstable regions commonly found within genomes that have consequences for evolution and disease. In humans, polymorphic TRs are known to cause neurodegenerative and neuromuscular disorders as well as being associated with complex diseases such as diabetes and cancer. If present in upstream regulatory regions, TRs can modify chromatin structure and affect transcription; resulting in altered gene expression and protein abundance. The most common TRs are short tandem repeats (STRs), or microsatellites. Promoter located STRs are considerably more polymorphic than coding region STRs. As such, they may be a common driver of phenotypic variation. To study STRs located in regulatory regions, we have performed genome-wide analysis to identify all STRs present in a region that is 2 kilobases upstream and 1 kilobase downstream of the transcription start sites of genes. Results: The Short Tandem Repeats in Regulatory Regions Table, STaRRRT, contains the results of the genome-wide analysis, outlining the characteristics of 5,264 STRs present in the upstream regulatory region of 4,441 human genes. Gene set enrichment analysis has revealed significant enrichment for STRs in cellular, transcriptional and neurological system gene promoters and genes important in ion and calcium homeostasis. The set of enriched terms has broad similarity to that seen in coding regions, suggesting that regulatory region STRs are subject to similar evolutionary pressures as STRs in coding regions and may, like coding region STRs, have an important role in controlling gene expression. Conclusions: STaRRRT is a readily-searchable resource for investigating potentially polymorphic STRs that could influence the expression of any gene of interest. The processes and genes enriched for regulatory region STRs provide potential novel targets for diagnosing and treating disease, and support a role for these STRs in the evolution of the human genome

Continuing difficulties in interpreting CNV data: lessons from a genome-wide CNV association study of Australian HNPCC/lynch syndrome patients

Background: Hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome (LS) is a cancer syndrome characterised by early-onset epithelial cancers, especially colorectal cancer (CRC) and endometrial cancer. The aim of the current study was to use SNP-array technology to identify genomic aberrations which could contribute to the increased risk of cancer in HNPCC/LS patients. Methods: Individuals diagnosed with HNPCC/LS (100) and healthy controls (384) were genotyped using the Illumina Human610-Quad SNP-arrays. Copy number variation (CNV) calling and association analyses were performed using Nexus software, with significant results validated using QuantiSNP. TaqMan Copy-Number assays were used for verification of CNVs showing significant association with HNPCC/LS identified by both software programs. Results: We detected copy number (CN) gains associated with HNPCC/LS status on chromosome 7q11.21 (28% cases and 0% controls, Nexus; p = 3.60E-20 and QuantiSNP; p < 1.00E-16) and 16p11.2 (46% in cases, while a CN loss was observed in 23% of controls, Nexus; p = 4.93E-21 and QuantiSNP; p = 5.00E-06) via in silico analyses. TaqMan Copy-Number assay was used for validation of CNVs showing significant association with HNPCC/LS. In addition, CNV burden (total CNV length, average CNV length and number of observed CNV events) was significantly greater in cases compared to controls. Conclusion: A greater CNV burden was identified in HNPCC/LS cases compared to controls supporting the notion of higher genomic instability in these patients. One intergenic locus on chromosome 7q11.21 is possibly associated with HNPCC/LS and deserves further investigation. The results from this study highlight the complexities of fluorescent based CNV analyses. The inefficiency of both CNV detection methods to reproducibly detect observed CNVs demonstrates the need for sequence data to be considered alongside intensity data to avoid false positive results

Non-Stimulated, Agonist-Stimulated and Store-Operated Ca²⁺ Influx in MDA-MB-468 Breast Cancer Cells and the Effect of EGF-Induced EMT on Calcium Entry

<div><p>In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca²⁺ reserves, molecular components of the store-operated Ca²⁺ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca²⁺ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca²⁺ influx. The potential roles for specific Ca²⁺ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca²⁺ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca²⁺ influx in a manner dependent on Ca²⁺ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca²⁺ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca²⁺-ATPase (time to peak [Ca²⁺]_CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); <em>P</em><0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca²⁺ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca²⁺ influx in non-stimulated cells.</p> </div

siRNA-mediated silencing of ORAI1 and TRPC1 in MDA-MB-468 breast cancer cells.

<p><b>A</b>) Quantitation of TRPC1 mRNA expression 48 h after treatment with TRPC1 siRNA (siTRPC1) relative to the non-targeting siRNA control (siNT). <b>B</b>) Assessment of ORAI1 expression 48 h post transfection with ORAI1 siRNA (siORAI1). Values show mean ± S.D. for six wells from three independent experiments; * <i>P</i><0.05 (unpaired t-test).</p

Effect of TRPC1 and ORAI1 silencing on constitutive ERK1/2 activity and cell proliferation.

<p><b>A</b>) Representative immunoblot showing constitutive phosphorylation and total expression of ERK1/2 in MDA-MB-468 cells with ORAI1 or TRPC1 silencing. <b>B</b>) Densitometric data was obtained from the pooled data (three independent immunoblots) and shows ERK1/2 phosphorylation relative to total ERK1/2 expression; * <i>P</i><0.05 (one-way ANOVA with Bonferroni post-tests). <b>C</b>) DAPI (cell number) and EdU staining (showing cells in S-phase of the cell cycle). <b>D</b>) Quantitation of the average cell count and (<b>E</b>) EdU positivity for nine wells from three independent experiments.* <i>P</i><0.05 (unpaired t-test). All graphs show mean ± S.D. Scale bar represents 100 µm.</p