Nucleoplasmic calcium regulates cell proliferation through legumain

Background & Aims: Nucleoplasmic Ca2+ 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 Ca2+ 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 Ca2+-binding sites inactivated. The subtraction permitted the selection of genes whose expression was affected by a small alteration in nuclear Ca2+ concentration. Results: The asparaginyl endopeptidase legumain (LGMN) was identified in this screening. When Ca2+ 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 SKHepl cells by similar to 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 Ca2+ signals regulate cell proliferation in part through the modulation of LGMN expression. Increased expression of LGMN may be involved in liver carcinogenesis. (C) 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.