title: Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.
abstract: BACKGROUND
  An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.
  
  
  RESULTS
  We utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.
  
  
  CONCLUSIONS
  Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Dragoni
authors.affiliation: Department of Molecular Medicine, University of Pavia, Pavia, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Laforenza
authors.affiliation: Centre for the Study of Myelofibrosis, Laboratory of Biotechnology, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bonetti
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Reforgiato
authors.affiliation: Centre for the Study of Myelofibrosis, Laboratory of Biotechnology, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Poletto
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lodola
authors.affiliation: Department of Molecular Medicine, University of Pavia, Pavia, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Bottino
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Guido
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Rappa
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Sumedha
authors.initials: S
authors.lastname: Pareek
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Tomasello
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Guarrera
authors.affiliation: Department of Public Health, University of Naples "Federico II", Naples, Italy.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Cinelli
authors.affiliation: Centre for the Study of Myelofibrosis, Laboratory of Biotechnology, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Aronica
authors.affiliation: Department of Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Germano
authors.initials: G
authors.lastname: Guerra
authors.affiliation: Centre for the Study of Myelofibrosis, Laboratory of Biotechnology, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Barosi
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Tanzi
authors.affiliation: Centre for the Study of Myelofibrosis, Laboratory of Biotechnology, Foundation IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Rosti
authors.affiliation: Laboratory of General Physioloy, Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Moccia
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000813:Anilides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D003114:Colony-Forming Units Assay; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D066026:Endothelial Progenitor Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005682:Gadolinium / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D055728:Primary Myelofibrosis / Q000235:genetics* / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013830:Thiadiazoles / Q000494:pharmacology; D055815:Young Adult
chemical_list: C476308:4-methyl-4'-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide; D000813:Anilides; D015220:Calcium Channels; D007211:Indoles; D008565:Membrane Proteins; D012333:RNA, Messenger; D050052:TRPC Cation Channels; D013830:Thiadiazoles; D007811:Lanthanum; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D005682:Gadolinium; D002118:Calcium; C000543:cyclopiazonic acid