The Cynthia Leveille-Webster Lab

Research Publications Cell Biology Physiology

 

Hepatocyte Cell Death and Hepatobiliary Disease 

The long range goal of the studies in our laboratory is to understand factors that fuel hepatocyte cell death and the mechanisms hepatocytes have evolved to survive these pathologic events.  Hepatocyte apoptosis accompanies congenital and acquired cholestatic hepatobiliary disorders and is due, in part, to the retention of endogenous compounds normally excreted in bile. Bile acids are among these retained toxins.  Since bile acids induce hepatocyte apoptosis/necrosis, it is likely they contribute to ongoing pathology in cholestatic liver disorders. The mechanism whereby bile acids induce hepatocyte cell death, however, is incompletely understood.

Hepatocytes possess mechanism s to defend against apoptosis. Increasing levels of the hormonal intracellular messanger, cAMP, is one means of defense. Cyclic AMP protects against bile acid as well as Fas ligand, tumor necrosis factor alpha, lipopolysaccharide and fatty acid induced hepatocyte apoptosis. We have shown that in vitro in rat hepatocyte cultures that the protective effect of cAMP in bile acid induced apoptosis is independent of protein kinase A (PKA), but instead proceeds through a cAMP mediated activation of cAMP-GEF’s (cAMP-regulated guanine nucleotide exchange factors, also known as EPAC, exchange proteins activated by cAMP). Protection proceeds through a Src tyrosine kinase/ phosphoinositide-3-kinase α/β (PI3K)/Akt /glycogen synthase kinase (GSK)3β signaling pathway (Figure 1).

Webster Fig 1

Figure 1. Putative cAMP-GEF survival signaling in hepatocytes.

Cyclic AMP-GEF’s Have Broad Cytoprotective Properties

There is substantial information in the literaturefrom our lab and that of others demonstrating that raising intracellular cAMP levels is not only cytoprotective for hepatobiliary cells, but can also protect cardiac myocytes, pancreatic beta cells, leukocytes and neurons from damage due to various insults. Furthermore, addition of cAMP to liver preservation solutions protects cell integrity in transplanted livers and emerging evidence suggests that cAMP-GEF pathways are also anti-fibrotic. Finally, it is well known that the diseased liver develops dysregulations in cAMP signaling that, if corrected, should promote hepatocyte health. Thus therapeutics targeted at cAMP-GEF survival signaling pathways could have broad applications in preserving hepatobiliary cell health. Our laboratory is involved in determining the molecular mechanism(s) by which the PI3K/Akt/GSK3β pathway protects hepatocytes from bile acid induced hepatocyte apoptosis.

Retained Bile Acids Can Induce Hepatocyte Cell Death

Bile acids are organic anions synthesized exclusively in the liver from cholesterol.  They are formed when a hydroxy group (OH) is added to the 7th position of cholesterol’s steroid nucleus. Since the cholesterol already has a 3-OH group, the simplest bile acids are the 3-, 7-di-OH bile acids such as chenodeoxycholate. Additional hydroxylation at the 12- position creates the tri-OH bile acids in the cholate group.  Bile acids are conjugated in the liver to either taurine or glycine.  In humans, the major circulating bile acid is the di-OH bile acid, glycochenodeoxycholate.  The major physiologic functions of bile acids are to stimulate hepatocyte bile flow and to promote intestinal fat absorption.

Numerous experimental studies have shown that at high concentrations bile acids are hepatotoxic.   Since serum and hepatic retention of bile acids accompanies cholestatic hepatobiliary disorders, bile acids likely play an important role in the pathologic progression of many chronic hepatopathies.  The mechanisms whereby bile acids damage hepatocytes are not fully understood. Bile acids stimulate both extrinsic death receptor (DR) and intrinsic mitochondrially mediated apoptosis.

Webster Fig 2 

Figure 2. Bile acid-induced apoptosis in cultured rat hepatocytes.

The extrinsic pathway DR pathway involves bile acid induced ligand independent oligomerization and activation of the Fas or TRAIL DR. DR receptor activation leads to the formation of a death initiating signaling complex (DISC) consisting of FADD and an initiator caspase, pro-caspase 8/10.  The initiator caspases are cleaved and activated within the DISC.  In Type I cells, enough pro-caspase 8/10 is cleaved to directly activate the effector caspases 3, 6 and 7.  Once activated these caspases carry out the demolition phase of apoptosis.  Most studies agree that hepatocytes are Type II cells and cannot generate enough active caspase 8/10 to cleave effector caspases.  Instead, they rely on a mitochondrial amplification loop in which pro-apoptotic initiator Bcl-2 molecules, such as Bid and Bim, translocate to the mitochondria and facilitate the activation of  pro-apoptotic effector Bcl-2 proteins, Bax and/or Bak.   Activation of Bax/Bak results in mitochondrial outer membrane permeabilization (MOMP) which commits the cells to die.  MOMP leads to the release of the intramembrane mitochondrial protein, cytochrome C, into the cytosol. Cytosolic cytochrome C stimulates the assembly of a signaling complex composed of Apaf-1, caspase 9, and ATP.  Caspase-9 is activated within the apoptosome and subsequently activates the effector caspases.

Intrinsic Apoptotic Pathway Signaling and Bile Acid Injury

The intrinsic apoptotic pathway shares its final cell death machinery with the DR mitochondrial amplification loop. Accumulating data support a role for intrinsic apoptosis in bile acid injury including: 1. the fact that hepatocytes from mice lacking DR’s or Bid still undergo death in vivo after bile duct ligation or in culture after exposure to bile acids, although cell death in both setting is attenuated. and 2. blockage of DR signaling with genetic inactivation of FADD fails to block bile acid induced apoptosis. We have data, as well, to show that FADD inhibition has no effect on bile acid induced apoptosis in rat hepatocytes.  The mechanism whereby bile acids activate intrinsic apoptosis is not well characterized and is one of the areas of focus in our laboratory.

Webster Fig 3 

Figure 3. Putative mechanisms of bile acid-induced intrinsic apoptosis.

Bile acids induce several known triggers of intrinsic apoptosis including ROS formation, activation of JNK and induction of ER stress. Bile acids also promote the mitochondrial membrane permeability transition (MMPT) which under the correct circumstances can trigger mitochondrial apoptosis.  Inhibition of all of these pathways can ameliorate bile acid cell death. We have evidence that bile acid activation of intrinsic apoptosis may involve a PI3Kγ/Rac pathway (see Figure 3).  We have shown that: 1. GCDC activates both the GTPase activity of Rac and the p110γ isoform of PI3K, 2. GCDC induced activation of Rac is sensitive to chemical inhibition of PI3Kγ and 3. chemical inhibition of Rac and PI3Kγ ameliorates GCDC induced apoptosis.  These observations are also compatible with recent studies which show Rac and PI3Kγ inhibition can thwart hepatocyte injury in vivo.  Investigations into the role of PI3Kγ and Rac in bile acid induced apoptosis are ongoing in our laboratory.

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