Cálcio intracelular na proliferação de células hepáticas

Abstract

Liver regeneration depends upon growth factors such as hepatic growth facto (HGF) and insulin that induce Ca2+ signals are necessary for proliferation of cells. Ca2+ signals in hepatocytes are mediated by inositol 1,4,5-triphosphate receptors (InP3Rs) activity, therefore, the mechanism by which nuclear Ca2+ regulates liver regeneration isnot known. Here we examined whether nuclear Ca2+ modulates expression of genes involved in cell proliferation and availed the contribution of InP3RI-mediated Ca2+ signaling to the regulation of liver regeneration. To investigate the role of nuclear Ca2+ in gene expression we used Rapid Subtraction Hybridization (RaSH) protocol thatpermit subtract genes expressed in SKHep1 cells with nuclear Ca2+ buffered. The subtraction permitted identification of 154 gens whose expression was affected by a small alteration in nuclear Ca2+ concentration. Among the selected clones, 3 clones waschoose, legumain (LGMN), reticulon 4 (RTN4) and transforming growth factor beta regulator 4 (TBRG4). These clones showed involvement in proliferative process by BLASTN analysis. The LGMN was chosen to investigate in details studies. We observed that buffering of nuclear Ca2+ reduced mRNA and protein LGMN levels. On the other hand, increases in nuclear Ca2+, by HGF stimulation, increased the LGMN expression. Silenciamento of LGMN by siRNA decreased proliferation of SKHep1cells, by a mechanism that not depends of endopeptidase activity of LGMN. The inhibitor of LGMN activity did not affect BrdU incorporation. Moreover, was observed that in LGMN absence there was reduction in mitotic index, with a significant reduction on fraction of cells in G2/M phase. This was associated with an increase in expressionof cyclins A and E and no apoptosis. The role of LGN in proliferation was confirmed in human tissue, where we observed increased expression of LGMN in hepatocellular carcinoma cells relative to normal hepatocytes in the same specimens. As the increase of intracellular Ca2+ in liver cells is mediated primarily by InsP3R, we investigated the relative contribution of isoform type I InsP3R in the proliferative capacity of hepatocytes during liver regeneration. We used siRNA coupled to the adenoviral systemto decreases the expression InsP3R both in vitro and in vivo. Adenoviruses AdsiRNA-I were effective and specific to reduce both receptor expression in CHO cells and in hepatocytes. The Ca2+ signaling was impaired in animals with InsP3RI silenced and this affected the process of liver regeneration. Together, these data show that the nuclearCa2+ regulates the process of proliferation of liver cells, at least in part by the xvi modulation of gene LGMN. Furthermore, we demonstrated that Ca2+ signaling mediated by InsP3RI is important for regulation of liver regeneration.