**4. Endoplasmic reticulum involvement in hydrophobic bile acid-induced apoptosis in rat hepatocytes**

Some Findings on Apoptosis in Hepatocytes 211

CHOP in vitro [51]. When hepatocytes of CHOP-null mice were incubated with toxic GCDCA, cell death was decreased [52]. In a hereditary model of intrahepatic cholestasis, accumulation of bile acids in the liver was correlated with ER stress [53]. We ourselves studied the effects of GCDCA treatment in hepatocytes, and found that when the culture medium contained Ca2+, persistent increase in intracellular Ca2+ was observed while only transient increase in intracellular Ca2+ was observed when cells were cultured in Ca2+ free medium, showing that GCDCA promotes Ca2+ influx from extracellular matrix and release of Ca2+ from ER. Activations of calpain and caspase-12 due to the increase in intracellular Ca2+ were also observed, and we reported that GCDCA induces apoptosis mediated by ER stress [29]. Furthermore, we reported the correlation between ER stress associated with GCDCA and caspase-8 activation. GCDCA activates caspase-8 via Fas death receptor, but when hepatocytes were pretreated with a caspase-8 inhibitor, z-IETD-FMK, expressions of BIP, an ER chaperone molecule, and CHOP, an ER stress response transcription factor, were suppressed. From these, we speculated that caspase-8 activated by GCDCA regulates ER

**5. Detailed mechanism of hydrophobic bile acid-induced apoptosis in** 

A hydrophobic bile acid GCDCA activates caspase-8 in hepatocytes via Fas death receptor in the cell membrane. Activated caspase-8 enhances mitochondrial membrane permeability to facilitate the release of cytochrome c, a pro-apoptotic protein, activates caspase-9 and caspase-3 to induce apoptosis. Caspase-8 activated by GCDCA cleaves BAP31 protein in ER membrane and may possibly be associated with Ca2+ release from mitochondria from ER. BAP31 is cleaved to be BAP20 which is an activated form. As shown in Fig. 3, intact (uncleaved) BAP31 protein is observed in ER in untreated hepatic cells, but in GCDCA-treated hepatic cells, the content of intact BAP31 protein is decreased. The decrease in intact BAP31 observed in GCDCA-treated hepatocytes was suppressed by pretreatment with z-IETD-FMK. When recombinant active caspase-8 was added to hepatocytes, decrease in intact BAP31 was observed. GCDCA-induced increased intracellular Ca2+ interacted with mitochondria and caused its dysfunction, followed by increase in mitochondrial membrane permeability and release of pro-apoptotic factors from mitochondria. Also, calpain was activated by the increase in Ca2+ release followed by activation of caspases. Furthermore, ER chaperone BIP is usually bound to an ER stress sensor located within the lumen of ER, but GCDCA treatment causes the increase in unfolded protein, and promotes the release of BIP from the stress sensor and increases the expression of CHOP, an ER stress-related transcription factor that works in the downstream of PERK, an ER stress sensor (Fig. 4). CHOP induces the transcription of Bim, a pro-apoptotic BH3 only protein [55]. Membranes of mitochondria and ER are connected

These complexes facilitate the transport of calcium and phospholipids and possibly are associated with apoptosis. GCDCA is known to induce apoptosis via mitochondria- and ER

stress [54].

**HepG2 cells** 

by complexes composed of tethering proteins [50].

In addition to the major two intracellular apoptotic pathways, that is, death receptormediated and mitochondria-mediated pathways, attention has recently been focused on the ER stress-mediated pathway. The response against the accumulation of unfolded proteins in ER is called unfolded protein response (UPR) and this is featured by activation of three different signaling pathways, inositol-requiring (IRE)-1�, protein kinase RNA-activated (PKR)-like ER kinase (PERK), activating transcription factor (ATF)-6�. Changes in ER functions are induced by various stimulations, extrinsic chemicals administered pharmacologically, or increase in physiologically secreted proteins, and these upsets are called ER stress, which can be detected by the UPR transducer activation. ER stress is observed in many liver diseases, and UPR activity is correlated with hepatic resistance against insulin in obesity and fatty liver. Chronic viral B and C hepatitis, alcohol-induced liver injury, ischemic re-perfusion damages, and cholestatic liver injury are also correlated with UPR activity. Prolonged or potent ER stress induces apoptosis.

Just like an anti-apoptotic protein Bcl-2, pro-apoptotic proteins Bax and Bak are localized in membranes of ER, and regulate homeostasis of Ca2+ within the cell. Release of Ca2+ from ER activates calpain, which in turn activates caspase-12 (in human cells, caspase-4), and initiates apoptosis. Ca2+ intake by mitochondria leads to enhancement of mitochondrial membrane permeability, and cytochrome c is released from mitochondria. Membranes of mitochondria and ER are connected through protein junctions [50]. These junctions seem to facilitate transports of Ca2+ and phospholipids, and are possibly associated with apoptosis. However, the exact signaling pathway that mediates ER stress-induced apoptosis in hepatocytes is not clarified yet. UPR is activated for restoration of ER homeostasis. The upset of ER homeostasis induced by damages or activations of UPR sensors is observed in some liver diseases, and ER stress is observed in various liver diseases. Drugs that are hepatotoxic activate several intracellular stress responses (for example, lysosome disorder, increased permeability of mitochondrial membrane, oxidative stress, inflammation and so on) in hepatocytes.

Correlations have been observed between these responses and ER dysfunction. Steatosis occurs in the liver after an acute ER stress mediated by a transcription factor for lipid regulation, sterol regulatory element-binding protein (SREBP)-1c and SREBP-2c. Activation of nuclear factor kappa B (NF-кB) occurs in the downstream of ER upset in alpha-1 antitrypsin (AAT) deficiency disease. Chronic viral hepatitis (hepatitis C virus HCV, hepatitis B virus HBV) accompanies ER dysfunction. Prolonged ER stress leads to apoptosis by activation of C/enhancer binding protein (EBP) homologous protein (CHOP), change in Ca2+ homeostasis and premature resuming of mRNA translation.

In cholestasis, toxic hydrophobic bile acid/salts are retained in the liver due to impaired biliary excretion. Sodium deoxycholate (DC) induced expressions of UPR genes BIP and CHOP in vitro [51]. When hepatocytes of CHOP-null mice were incubated with toxic GCDCA, cell death was decreased [52]. In a hereditary model of intrahepatic cholestasis, accumulation of bile acids in the liver was correlated with ER stress [53]. We ourselves studied the effects of GCDCA treatment in hepatocytes, and found that when the culture medium contained Ca2+, persistent increase in intracellular Ca2+ was observed while only transient increase in intracellular Ca2+ was observed when cells were cultured in Ca2+ free medium, showing that GCDCA promotes Ca2+ influx from extracellular matrix and release of Ca2+ from ER. Activations of calpain and caspase-12 due to the increase in intracellular Ca2+ were also observed, and we reported that GCDCA induces apoptosis mediated by ER stress [29]. Furthermore, we reported the correlation between ER stress associated with GCDCA and caspase-8 activation. GCDCA activates caspase-8 via Fas death receptor, but when hepatocytes were pretreated with a caspase-8 inhibitor, z-IETD-FMK, expressions of BIP, an ER chaperone molecule, and CHOP, an ER stress response transcription factor, were suppressed. From these, we speculated that caspase-8 activated by GCDCA regulates ER stress [54].

210 Apoptosis and Medicine

hepatocytes.

**apoptosis in rat hepatocytes** 

**4. Endoplasmic reticulum involvement in hydrophobic bile acid-induced** 

In addition to the major two intracellular apoptotic pathways, that is, death receptormediated and mitochondria-mediated pathways, attention has recently been focused on the ER stress-mediated pathway. The response against the accumulation of unfolded proteins in ER is called unfolded protein response (UPR) and this is featured by activation of three different signaling pathways, inositol-requiring (IRE)-1�, protein kinase RNA-activated (PKR)-like ER kinase (PERK), activating transcription factor (ATF)-6�. Changes in ER functions are induced by various stimulations, extrinsic chemicals administered pharmacologically, or increase in physiologically secreted proteins, and these upsets are called ER stress, which can be detected by the UPR transducer activation. ER stress is observed in many liver diseases, and UPR activity is correlated with hepatic resistance against insulin in obesity and fatty liver. Chronic viral B and C hepatitis, alcohol-induced liver injury, ischemic re-perfusion damages, and cholestatic liver injury are also correlated

Just like an anti-apoptotic protein Bcl-2, pro-apoptotic proteins Bax and Bak are localized in membranes of ER, and regulate homeostasis of Ca2+ within the cell. Release of Ca2+ from ER activates calpain, which in turn activates caspase-12 (in human cells, caspase-4), and initiates apoptosis. Ca2+ intake by mitochondria leads to enhancement of mitochondrial membrane permeability, and cytochrome c is released from mitochondria. Membranes of mitochondria and ER are connected through protein junctions [50]. These junctions seem to facilitate transports of Ca2+ and phospholipids, and are possibly associated with apoptosis. However, the exact signaling pathway that mediates ER stress-induced apoptosis in hepatocytes is not clarified yet. UPR is activated for restoration of ER homeostasis. The upset of ER homeostasis induced by damages or activations of UPR sensors is observed in some liver diseases, and ER stress is observed in various liver diseases. Drugs that are hepatotoxic activate several intracellular stress responses (for example, lysosome disorder, increased permeability of mitochondrial membrane, oxidative stress, inflammation and so on) in

Correlations have been observed between these responses and ER dysfunction. Steatosis occurs in the liver after an acute ER stress mediated by a transcription factor for lipid regulation, sterol regulatory element-binding protein (SREBP)-1c and SREBP-2c. Activation of nuclear factor kappa B (NF-кB) occurs in the downstream of ER upset in alpha-1 antitrypsin (AAT) deficiency disease. Chronic viral hepatitis (hepatitis C virus HCV, hepatitis B virus HBV) accompanies ER dysfunction. Prolonged ER stress leads to apoptosis by activation of C/enhancer binding protein (EBP) homologous protein (CHOP), change in

In cholestasis, toxic hydrophobic bile acid/salts are retained in the liver due to impaired biliary excretion. Sodium deoxycholate (DC) induced expressions of UPR genes BIP and

with UPR activity. Prolonged or potent ER stress induces apoptosis.

Ca2+ homeostasis and premature resuming of mRNA translation.
