Guangsheng Fan

A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.

Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation

The hydrophilic bile salt ursodeoxycholic acid (UDCA) is a potent inhibitor of apoptosis. In this paper, we further characterize the mechanism by which UDCA inhibits apoptosis induced by deoxycholic acid, okadaic acid and transforming growth factor β1 in primary rat hepatocytes. Our data indicate that coincubation of cells with UDCA and each of the apoptosis-inducing agents was associated with an approximately 80% inhibition of nuclear fragmentation (P<0.001). Moreover, UDCA prevented mitochondrial release of cytochrome c into the cytoplasm by 70–75% (P<0.001), thereby, inhibiting subsequent activation of DEVD-specific caspases and cleavage of poly(ADP-ribose) polymerase. Each of the apoptosis-inducing agents decreased mitochondrial transmembrane potential and increased mitochondrial-associated Bax protein levels. Coincubation with UDCA was associated with significant inhibition of these mitochondrial membrane alterations. The results suggest that the mechanism by which UDCA inhibits apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited.

p53 dephosphorylation and p21Cip1/Waf1 translocation correlate with caspase-3 activation in TGF-β1-induced apoptosis of HuH-7 cells

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor β1 (TGF-β1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-β1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-β1. The results indicated that greater than 90% of the TGF-β1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21Cip1/Waf1 expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21Cip1/Waf1 and p27Kip1 into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-β1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-β1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21Cip1/Waf1 and p27Kip1, resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-β1-induced apoptosis downstream of the pRb/E2F-1 pathway.

Molecular Regulation of Liver Regenerationa

The liver constitutes one of the few, normally quiescent tissues in the adult animal that has the capacity to regenerate in response to cell loss through physical, infectious or hepatotoxic injury. The most popular experimental model to study hepatic regeneration was reported by Higgins and Anderson in 1931 in which they described the surgical removal of two-thirds of the liver in rats.’ In the remaining lobes, the majority of hepatic cells rapidly reenter the growth cycle and begin to replicate. The “regeneration” of the liver after 70% partial hepatectomy (PH) is a precise, highly regulated process which appears to be controlled by many of the same factors responsible for the liver’s fetal development. Restoration of liver mass is governed by functional rather than anatomical factors, occurs by compensatory hyperplasia of the remnant tissue and, therefore, does not represent true regeneration. In the paradigm of “hepatic regeneration” after PH, liver function recovers quickly following the restoration of histologically normal tissue. The liver appears to have an optimal functional mass and as the remnant tissue grows, its shape is largely determined by external pressure. Reorganization of lobular architecture is a dynamic process involving both dissolution and deposition of extracellular matrix.* The kinetics of cell replication displayed by hepatocytes after PH are well described and, in general, represent a fairly synchronized p r o ~ e s s . ~ ~ The exact timing of DNA synthesis after PH varies with the age of the animal and can be modified by hormones and dietary manipulations. For younger adult rats, the basal rate of DNA synthesis is unchanged in the first 12 hours (prereplicative phase) after which there is a wave of DNA synthesis in hepatocytes which peaks at about 24 hours and then gradually declines. Hepatocytes located in the periportal area

A Novel Link Between REC2, a DNA Recombinase, the Retinoblastoma Protein, and Apoptosis

The REC2 recombinase is essential for recombinational repair following DNA damage as well as for successful meiosis and gene targeting in the corn smut Ustilago maydis. Here we report that overexpression of REC2 induced apoptotic cell death in human HuH-7, Hep G2, and Hep 3B hepatoma cells. Apoptosis was related to recombinase activity and was significantly increased by inhibition of retinoblastoma (Rb) expression with transforming growth factor-β1. REC2-induced apoptosis was associated with a significantly reduced percentage of cells in the G1phase of the cell cycle and a significant reduction in G2/M only in theRb( − / − )Hep 3B cells. Overexpression of REC2 resulted in increased abundance of the hyperphosphorylated form of Rb. However, by immunoprecipitation REC2 was associated primarily with hypophosphorylated Rb, suggesting that REC2 may be involved in modulating the phosphorylation state of Rb. The A and B pocket domains with the spacer amino acid sequence and the carboxyl-terminal region of Rb were required for maximal binding to REC2. Overexpression of Rb significantly inhibited REC2-induced apoptosis even in the presence of transforming growth factor-β1. Taken together, these data suggest a novel interaction of Rb with the recombinase REC2 and a role for this complex in bridging DNA recombination and apoptosis.