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Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line
B Jones1, P J Roberts, W A Faubion
1Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.
Abstract:
We have previously demonstrated abrogation of bile salt-induced apoptosis by cathepsin B inhibitors. However, caspases have been strongly implicated in apoptosis, and the mechanistic interface between caspase and cathepsin B activation is unclear. Thus our aims were to determine the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis in a rat hepatoma cell line. Expression of cystatin A was used to inhibit cathepsin B, whereas Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used to inhibit caspases. Cystatin A expression prevented cathepsin B activation and apoptosis during treatment with glycochenodeoxycholate (GCDC), a toxic bile salt. Caspase N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolytic activity increased in both wild-type and cystatin A-transfected cells treated with GCDC, demonstrating caspase activation despite inhibition of cathepsin B. In contrast, Z-VAD-FMK blocked both DEVD-AMC hydrolytic activity and cathepsin B activity during GCDC treatment. Our data demonstrate that 1) bile salt-induced apoptosis can be inhibited by the cystatin A transgene and 2) caspase and cathepsin B activation are linked mechanistically with cathepsin B downstream of caspases.
Insights
Bile salt-induced apoptosis involves both caspases and cathepsin B. Caspase activation precedes and triggers cathepsin B activation, revealing a mechanistic link in this cell death pathway.
Area of Science:
- Hepatology
- Cell Biology
- Biochemistry
Background:
- Bile salt-induced apoptosis is a significant cellular process.
- The roles of caspases and cathepsin B in this pathway are established, but their mechanistic relationship remains unclear.
Purpose of the Study:
- To elucidate the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis.
- To investigate the sequential activation of these proteases in a rat hepatoma cell line.
Main Methods:
- Utilized cystatin A expression to inhibit cathepsin B.
- Employed Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) to inhibit caspases.
- Measured caspase activity using N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolysis.
Main Results:
- Cystatin A expression inhibited cathepsin B activation and apoptosis induced by glycochenodeoxycholate (GCDC).
- GCDC treatment increased caspase activity even when cathepsin B was inhibited.
- Z-VAD-FMK treatment blocked both caspase and cathepsin B activity during GCDC exposure.
Conclusions:
- Bile salt-induced apoptosis can be effectively inhibited by cystatin A.
- Caspase activation mechanistically precedes and triggers cathepsin B activation in this apoptotic pathway.