The principal findings of this study relate Ctsb inactivation to liver injury and fibrogenesis in cholestasis. The observations demonstrate that, in the BDL animal, both genetic and pharmacologic inactivation of Ctsb reduces (a) hepatocyte apoptosis, serum ALT values, and histologic evidence of liver injury; (b) hepatic inflammation, as assessed by expression of chemoattractants and neutrophil infiltration; (c) mRNA expression for markers of HSC fibrogenic activity; and (d) collagen deposition. Taken together, these observations suggest a critical role for this protease in mediating the deleterious consequences of cholestasis. Each of these observations is discussed in greater detail below.
Increasing evidence implicates Ctsb as a proapoptotic protease. For example, Ctsb contributes to bile salt– and TNF-α–induced hepatocyte apoptosis (10
), and pharmacologic inhibition of Ctsb blocks apoptosis induced by p53 and cytotoxic agents (32
). Ctsb activity appears to play a critical role in lysosomal permeabilization in cytotoxic events, causing release of lysosomal proteases into the cytosol, which in turn cause mitochondrial dysfunction (10
). The current findings extend these observations by demonstrating a role for this protease in vivo during a model of human liver disease, namely, cholestasis. Consistent with the prior in vitro studies, Ctsb would appear to contribute to hepatocyte apoptosis by causing mitochondrial dysfunction and cytochrome c
release. Indeed, cytosolic cytochrome c
was markedly reduced in BDL Ctsb–/–
and in R-3032–treated Ctsb+/+
animals compared with BDL WT animals. How Ctsb contributes to mitochondrial dysfunction remains to be elucidated, but our previous in vitro studies implicated a requirement for additional cytosolic factor(s) (10
). Nonetheless, the current studies provide mechanistic evidence implicating the lysosomal protease Ctsb and therefore a dominant role for the lysosomal pathway of hepatocyte apoptosis in cholestatic liver injury.
Our study also shows that Ctsb-mediated liver injury not only causes apoptosis but also stimulates production of proinflammatory chemokines. Hepatocyte apoptosis therefore appears to be associated with the generation of chemokines promoting inflammation in cholestasis. Indeed, in other models of liver injury hepatocyte apoptosis has also been linked to inflammation. For example, Fas-mediated hepatocyte apoptosis elicits an inflammatory response in the liver that could secondarily induce HSC activation (25
). Although originally thought of as a “silent process,” unorchestrated and continuous apoptosis in the liver is likely profibrogenic in pathophysiologic conditions.
Our studies suggest a role for Ctsb in hepatic fibrogenesis during cholestasis. Ctsb could have a direct or indirect effect on hepatic fibrogenesis. It is likely that Ctsb inactivation and consequent attenuation of hepatocyte apoptosis is the mechanism linking this proapoptotic protease to liver fibrogenesis. Indeed, inhibition of Fas-mediated apoptosis also attenuates hepatic fibrogenesis, supporting a link between liver cell apoptosis and HSC activation (18
). The mechanism by which hepatocyte apoptosis results in hepatic fibrogenesis is likely indirect. Hepatocyte apoptosis elicits an inflammatory response associated with chemokine expression and neutrophil infiltration (5
). This inflammatory response has been well established to cause stellate cell activation (34
). The activated stellate cells produce collagen, causing liver scarring. Alternatively, engulfment of hepatocyte apoptotic bodies directly by stellate cells may also promote activation of these cells as has been observed in vitro (35
). However, based on current concepts, we favor the following model linking Ctsb-associated hepatocyte apoptosis to inflammation and stellate cell activation. Ctsb, via cytotoxic signaling cascades initiated by toxic bile acids or perhaps TNF-α, induces hepatocyte apoptosis. This injurious process promotes hepatic inflammatory responses. The inflammation subsequently induces stellate cell activation and fibrogenesis. This linear model (apoptosis → inflammation → fibrosis) is likely simplistic but provides a framework to further study apoptosis as an inciting event in liver injury and scarring.
In summary, our findings suggest that during extrahepatic cholestasis in the mouse, liver injury, inflammation, markers of stellate cell activation, and elevation of indices of hepatic fibrogenesis are, in part, Ctsb dependent. Furthermore, inactivation of Ctsb catalytic activity with selective protease inhibitors such as R-3032 indicates a potential therapeutic option for cholestatic liver injury, inflammation, and fibrosis. These data also implicate a mechanistic link between hepatocyte apoptosis and HSC activation and suggest inhibition of apoptosis would diminish liver fibrosis in chronic cholestatic liver diseases. These concepts merit further investigation as mechanism(s) contributing to the development of cirrhosis and as potential antifibrotic therapeutic strategies.