Viviane Andrade1, Mateus Guerra2, Camila Jardim3, Flavia Melo3, Wamberto Silva4, Jose M Ortega1, Marie Robert5, Michael H Nathanson6, Fatima Leite3. 1. Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 2. 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. 3. Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 4. Department of Genetics, Center for Cell Based Therapy, University of São Paulo, Ribeirão Preto, SP, Brazil. 5. Department of Pathology, Yale University School of Medicine, New Haven, CT, USA. 6. 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.
Abstract
BACKGROUND & AIMS: 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.
BACKGROUND & AIMS: 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 endopeptidaselegumain (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: M J Hendzel; Y Wei; M A Mancini; A Van Hooser; T Ranalli; B R Brinkley; D P Bazett-Jones; C D Allis Journal: Chromosoma Date: 1997-11 Impact factor: 4.316
Authors: Raghavendra Vasudeva Murthy; Gunnar Arbman; Jingfang Gao; G David Roodman; Xiao-Feng Sun Journal: Clin Cancer Res Date: 2005-03-15 Impact factor: 12.531
Authors: Chaido Stathopoulou; Arunakumar Gangaplara; Grace Mallett; Francis A Flomerfelt; Lukasz P Liniany; David Knight; Leigh A Samsel; Rolando Berlinguer-Palmini; Joshua J Yim; Tania C Felizardo; Michael A Eckhaus; Laura Edgington-Mitchell; Jonathan Martinez-Fabregas; Jinfang Zhu; Daniel H Fowler; Sander I van Kasteren; Arian Laurence; Matthew Bogyo; Colin Watts; Ethan M Shevach; Shoba Amarnath Journal: Immunity Date: 2018-07-24 Impact factor: 31.745