title: Nucleoplasmic calcium regulates cell proliferation through legumain.
abstract: OBJECTIVE
  Nucleoplasmic Ca(2+) regulates cell growth in the liver, but the proteins through which this occurs are unknown.
  
  
  METHODS
  We used Rapid Subtraction Hybridization (RaSH) to subtract genes in SKHep1 liver cells expressing the Ca(2+) buffer protein parvalbumin (PV) targeted to the nucleus, from genes in cells expressing a mutated form of nuclear-targeted PV which has one of two Ca(2+)-binding sites inactivated. The subtraction permitted the selection of genes whose expression was affected by a small alteration in nuclear Ca(2+) concentration.
  
  
  RESULTS
  The asparaginyl endopeptidase legumain (LGMN) was identified in this screening. When Ca(2+) was buffered in the nucleus of SKHep1 cells, LGMN mRNA was decreased by 97%, in part by a transcriptional mechanism, and decreased expression at the protein level was observed by immunoblot and immunofluorescence. Treatment with hepatocyte growth factor increased LGMN expression. Knockdown of LGMN by siRNA decreased proliferation of SKHep1 cells by ∼50% as measured both by BrdU uptake and mitotic index, although an inhibitor of LGMN activity did not affect BrdU incorporation. A significant reduction in the fraction of cells in G2/M phase was seen as well. This was associated with increases in the expression of cyclins A and E. Furthermore, LGMN expression was increased in hepatocellular carcinoma cells relative to normal hepatocytes in the same specimens.
  
  
  CONCLUSIONS
  These findings suggest a new role for LGMN and provide evidence that nuclear Ca(2+) signals regulate cell proliferation in part through the modulation of LGMN expression. Increased expression of LGMN may be involved in liver carcinogenesis.
authors.affiliation: Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Viviane
authors.initials: V
authors.lastname: Andrade
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Mateus
authors.initials: M
authors.lastname: Guerra
authors.affiliation: Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Camila
authors.initials: C
authors.lastname: Jardim
authors.affiliation: Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Flavia
authors.initials: F
authors.lastname: Melo
authors.affiliation: Department of Genetics, Center for Cell Based Therapy, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Wamberto
authors.initials: W
authors.lastname: Silva
authors.affiliation: Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Ortega
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Marie
authors.initials: M
authors.lastname: Robert
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. Electronic address: michael.nathanson@yale.edu.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Nathanson
authors.affiliation: Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
authors.forename: Fatima
authors.initials: F
authors.lastname: Leite
mesh_terms: D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D059565:G2 Phase Cell Cycle Checkpoints; D055785:Gene Knockdown Techniques; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000099240:Asparaginyl Endopeptidase
chemical_list: D002118:Calcium; D003546:Cysteine Endopeptidases; D012333:RNA, Messenger; D000099240:Asparaginyl Endopeptidase