LPS fromEscherichia coli0127: B8 and GalN were purchased from Sigma-Aldrich

LPS fromEscherichia coli0127: B8 and GalN were purchased from Sigma-Aldrich. == Experimental Style and Biochemical Analysis == Wild type and PXR-null mice with a mixed background of C57/BL/6 and SVJ129 were kindly provided by Dr. LPS/GalN-induced liver injury. Lack of PXR resulted in a significant reduction of LC3B-I, -II as well as Beclin-1 protein levels after LPS/GalN treatment. Taken together, PXR is usually a critical hepatoprotective factor. Increases of LPS/GalN-induced hepatocyte apoptosis and liver injury in PXR-null mice are due to deregulated MAP kinase activation as well as delayed Jak2/Stat3 activation, which lead to a compromise in defense mechanisms that involve Bcl-xL-, HO-1, and autophagy-mediated pathways. Keywords:Apoptosis, Jak2/Stat3, Liver Injury, MAP kinases, PXR, nuclear receptor The binding of TNF to TNFR1 induces rapid activation of several intracellular signaling pathways that influence cell survival and death (1). Among them, nuclear factor B (NF-B) is the major transcription factor that regulates anti-apoptotic proteins and prevents cells from TNF-induced apoptosis (1,2). Deletion of p65RelA, a key player of NFB signaling, causes severe hepatocyte apoptosis and embryonic lethality due to defective NFB signaling and uncontrolled TNF toxicity (3). The Stat3 transcription factor also plays an essential role in cell growth and apoptosis. Mice with specifically blocked hepatocyte Stat3 activation have increased apoptosis and liver injury induced by lipopolysaccharide (LPS), suggesting that Stat3-mediated survival signaling protects hepatocytes from TNF-induced apoptosis (4). In addition, activation of Stat3 blocks toll like receptor-mediated inflammation (5). TNF can induce mitogen-activated protein kinases (MAP kinases), including JNK1/2, MLT-748 ERK1/2, and p38, which transmit extracellular signals into their intracellular targets to regulate cell proliferation, differentiation, and apoptosis (1). Activation of MAP kinases has dual functions and presents both pro-apoptotic and anti-apoptotic functions in TNF-mediated cell death events. JNK knockout mice are resistant to TNF-induced liver injury indicating JNK mediates cell death signaling and promotes liver injury (6). In general, ERK signaling has been considered to mediate cell survival; however, increasing evidence demonstrates that ERK pathway also mediates apoptosis and tissue damage induced by different stimuli in different tissues (79). Depending on cell type or model of damage, inhibition of p38 can enhance (10) or prevent (11) TNF-induced apoptosis, thus the role of p38 in TNF-induced cell or tissue injury remains controversial. Nuclear receptor pregnane x receptor (PXR) regulates a range of genes associated with clearance of endobiotics and xenobiotics. Recent research has revealed the important role of PXR in intestine injury and inflammatory processes (1214). Crosstalk between PXR and NFB has been reported (12). Usingin vitromodels, it has been implicated that activated human PXR (hPXR) suppresses NFB MLT-748 and its target genes in primary human hepatocytes and intestinal LS180 cells (12). Activation of rodent PXR ameliorates dextran sulfate sodium-induced MLT-748 colitis through repression of NF-B target genes (13). Notably, activated PXR also directly up-regulates anti-apoptotic proteins such as Bcl-xL and Bcl-2 in cultured hepatocytes (15) and colon cancer cells (16). Activation of PXR inhibits human hepatic stellate cell trans-differentiationin vitro(17) and in carbon tetrachloride-induced liver fibrosis mouse model (18). PXR also provides neuroprotection in a mouse model of Niemann-Pick C disease (19). Despite emerging reports about the regulatory role of PXR in many physiological and pathological conditions, little is known about whether PXR regulates acute hepatic injuryin vivoand whether survival or pro-apoptotic signaling pathways are affected due to lack of PXR. LPS/D-galactosamine (GalN) treatment of mice is usually a classic model of acute liver injury. GalN is usually a specific hepatotoxic transcriptional inhibitor that abolishes protection of NFB and sensitizes liver towards subtoxic amounts of LPS (20) and TNF is the predominant mediator that induces hepatocyte apoptosis and liver injury (21,22). Our study examines thein vivorole of PXR in LPS/GalN-induced acute liver injury using wild type and PXR-null mice. We specifically decided thein vivoimpact of PXR on LPS/GalN-induced activation of hepatic MAP kinases and Stat3 as well as the downstream survival and death pathways. Our results are the PTCH1 first to demonstrate that loss of PXR potentiates LPS/GalN-induced hepatocyte apoptosis and liver injury. The enhanced liver injury found in PXR-null mice is usually associated with delayed activation of hepatic Stat3, deregulated phosphorylaton MLT-748 of MAP kinases, and MLT-748 a compromise in anti-apoptotic signaling. == MATERALS AND METHODS == == Reagents == Antibodies specific for phospho-Akt (Thr308), pan-Akt; phospho-Stat3 (Tyr705), phospho-Stat3 (Ser727), Stat3; phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK; phospho-p38 MAP kinase (Thr180/Tyr182), p38 MAP kinase; phospho-ERK1/2 MAP kinase (Thr202/Tyr204), ERK1/2 MAP kinase; phospho-Jak1 (Tyr1022/1023); phospho-Tyk2 (Tyr1054/1055); phospho-Jak2 (Tyr1007/1008), Jak2; cleaved caspase-3 (Asp175), Bcl-xL and Bax were purchased from Cell Signaling Technology. Antibody specific for phosphor-p65 (Ser311), HO-1 were.