Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1–induced apoptosis in rat hepatocytes

✍ Scribed by Susana Solá; Joana D. Amaral; Rui E. Castro; Rita M. Ramalho; Pedro M. Borralho; Betsy T. Kren; Hirotoshi Tanaka; Cifford J. Steer; Cecília M. P. Rodrigues

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 322 KB
• Volume: 42
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.20870

✦ Synopsis

Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor ␤1 (TGF-␤1)-induced hepatocyte apoptosis by a nuclear steroid receptor (NSR)-dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-␤1-induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-␤1-induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-␤1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925-934.) U rsodeoxycholic acid (UDCA) is widely used in the treatment of cholestatic liver diseases. Its therapeutic effects have been attributed to several mechanisms, 1 including modulation of classic mito-chondrial pathways of apoptosis. [2][3][4] Nevertheless, reduction of cell death by UDCA may also involve alternate and upstream molecular targets of the E2F-1/Mdm-2/p53 apoptotic pathway 5 mediated through nuclear steroid receptors (NSRs). 6 Transforming growth factor ␤1 (TGF-␤1) is a multifunctional cytokine that induces growth arrest and apoptosis in hepatic cells, in part through the E2F-1 transcription factor. 7,8 Our previous studies indicated that UDCA modulates the expression of apoptotic target genes induced by TGF-␤1 by increasing both expression and nuclear translocation of the glucocorticoid (GR) and the mineralocorticoid (MR) receptors. 6 The activation of NSR is modulated in vivo by compounds that interact directly with either the receptor moiety or its associated proteins. 9 UDCA is a cholesterolderived molecule, suggesting a possible interaction between this bile acid and NSR. In fact, a number of bile acids bind and inactivate the farnesoid X-activated receptor (FXR). 10,11 Although UDCA itself does not bind FXR, 10 it inhibits its activation by more hydrophobic bile acids. 12 Interestingly, other studies have demonstrated that UDCA interacts with GR. 13 The bile acid was shown