title: Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration.
abstract: BACKGROUND
  The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared.
  
  
  METHODS
  Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system.
  
  
  RESULTS
  CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space.
  
  
  CONCLUSIONS
  We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.
authors.affiliation: Unit of Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700, Fribourg, Switzerland.
authors.forename: Walter
authors.initials: W
authors.lastname: Blum
authors.affiliation: Unit of Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700, Fribourg, Switzerland.
authors.forename: László
authors.initials: L
authors.lastname: Pecze
authors.affiliation: Laboratory of Molecular Oncology, University Hospital Zürich, Labor 40E, Sternwartstrasse 14, 8091, Zurich, Switzerland.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Felley-Bosco
authors.affiliation: Unit of Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700, Fribourg, Switzerland. Beat.Schwaller@unifr.ch.
authors.forename: Beat
authors.initials: B
authors.lastname: Schwaller
mesh_terms: D000818:Animals; D064032:Calbindin 2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005838:Genotype; D000090204:Mesothelin; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010537:Peritoneum / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D061251:Primary Cell Culture; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
chemical_list: C576764:CALB2 protein, human; C576511:Calb2 protein, mouse; D064032:Calbindin 2; C000715528:Msln protein, mouse; D000090204:Mesothelin