Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-beta1-induced apoptosis in rat hepatocytes.
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 beta1 (TGF-beta1)-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 anti-apoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-beta1-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-beta1-induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-beta1. Finally, using a fluorescently 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 anti-apoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis.
Solá
Susana
S
Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.
Amaral
Joana D
JD
Castro
Rui E
RE
Ramalho
Rita M
RM
Borralho
Pedro M
PM
Kren
Betsy T
BT
Tanaka
Hirotoshi
H
Steer
Cifford J
CJ
Rodrigues
Cecília M P
CM
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D015870:Gene Expression; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014580:Ursodeoxycholic Acid / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014580:Ursodeoxycholic Acid