Retinoid-induced apoptosis and proliferation of hepatocytes are mediated by distinct nuclear receptors.

摘要

Retinoids, derivatives of vitamin A, are important signaling molecules involved in the regulation of cellular homeostasis including differentiation, apoptosis, and proliferation. The first part of this dissertation is focused on the mechanism underlying the differential susceptibilities of human hepatocellular carcinoma (HCC) cells to the apoptotic effect of a synthetic retinoid, fenretinide. The second part of this dissertation aims to investigate the mechanism responsible for 13-cis retinoic acid-induced hepatocyte proliferation.;Retinoids are used clinically to treat several types of cancer; however, their effects on liver cancer have not been fully characterized. To investigate the therapeutic potential of retinoids on HCC, the apoptotic effect of a panel of retinoids on three human HCC cell lines was evaluated and underlying mechanisms were investigated. Our results reveal that fenretinide effectively induced apoptosis in Huh-7 and Hep3B cells but not in HepG2 cells. Gene expression analysis of a panel of nuclear receptors demonstrates that the basal and inducibility of retinoic acid receptor (RAR) beta expression positively correlate with the susceptibilities of HCC cells to fenretinide, thus suggesting a role of RARbeta in this molecular determining process. Furthermore, transactivation of the RXRalpha/RARbeta-mediated pathway in the reporter gene assay establishes fenretinide as an effective ligand. Fenretinide also increases the binding of RARbeta to its cognate DNA response element (RARE, DR5) as demonstrated by chromatin immunoprecipitation (ChIP) assay. Knockdown of the endogenous RARbeta expression with siRNA markedly impairs fenretinide-induced apoptosis in the sensitive Huh-7 cells. Therefore, these results demonstrate that fenretinide activates RARbeta and induces RARbeta-dependent apoptosis in sensitive Huh-7 cells. These findings suggest a novel role for RARbeta as a tumor suppressor by mediating the signals of certain chemotherapeutic agents.;Besides RARbeta, one orphan nuclear receptor, Nur77, also seems to contribute to the differential sensitivities of human HCC cells to fenretinide-induced apoptosis. Our results clearly show that the expression of Nur77 positively correlates with the sensitivities of HCC cells to fenretinide. Moreover, the subcellular distribution patterns of Nur77 between Huh-7 and HepG2 cells in response to fenretinide were characterized and compared. It appears that Nur77 mediates the apoptotic effect of fenretinide in sensitive Huh-7 cells via actively targeting mitochondria whereas it confers HepG2 cells resistance by specifically accumulating in the nucleus. In addition, Nur77 knockdown by siRNA in Huh-7 cells demonstrates that the proapoptotic function of Nur77 was required for the full execution of fenretinide-induced apoptosis. Therefore, Nur77 exerts opposing effects in HCC cells in response to fenretinide, which is, at least in part, responsible for the observed differential susceptibilities of HCC cells.;Although retinoids cause growth arrest and differentiation in various cancer cells, they are also known to cause hepatomegaly in humans and rodents. Thus, the mechanism by which retinoids induce hepatocyte proliferation was examined. 13-cis retinoic acid (13-cis RA) stimulates proliferation of Hep3B cells, concomitant with elevation in DNA synthesis and cell cycle progression. In vivo, 13-cis RA increases hepatic DNA synthesis after 5- and 10-day treatments. The high expression ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II (CRABP II) in Hep3B cells and in mouse livers prior to 13-cis RA treatment suggests preferred activation of peroxisome proliferator activated receptor beta (PPARbeta) over retinoic acid receptor (RAR) by 13-cis RA. The ratio of FABP5/CRABP II is further increased after 13-cis RA treatment. Moreover, 13-cis RA activates PPARbeta and induces the expression of PPARbeta target genes including phosphoinositide-dependent protein kinase 1 (PDK-1) in Hep3B cells and mouse livers. Following induction of PDK-1, Akt is phosphorylated and activated in both Hep3B cells and mouse livers. Over-expression of PPARbeta in Hep3B cells further enhances 13-cis RA-induced DNA synthesis. Akt inhibitor blocks 13-cis RA-induced Akt activation and proliferation in a dose-dependent manner, but does not interfere with induction of PPARbeta target genes indicating PPARbeta is upstream of Akt. Activation of PPARbeta/PDK-1/Akt cascade is thus responsible for the proliferative effect of 13-cis RA. Therefore, 13-cis RA promotes hepatocyte proliferation through activation of PPARbeta.