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Background/Aims: The effect of CD95 ligand (CD95L) and cycloheximide (CHX) on CD95 membrane targeting and apoptosis was studied in activated, cultured rat hepatic stellate cells (HSC). Methods: CD95 membrane targeting was analysed by fluorescence staining. Protein and mRNA expression were determined by Western blotting and real time PCR. Apoptosis was detected by TUNEL, caspase-3 and -8 assay. Results: Neither CD95L nor CHX alone induced CD95 membrane trafficking and HSC apoptosis. When, however, both compounds were added simultaneously, CD95 was targeted within one hour to the plasma membrane and apoptosis was induced. CHX induced a transient and CD95L a delayed activation of c-Jun-N-terminal kinase (JNK), but when added together initial JNK activation was enhanced and prolonged. Inhibition of JNK abolished the CD95 membrane targeting in response to CD95L/CHX, however had little effect on the apoptotic response. Likewise, 8-CPT-cAMP inhibited CD95 membrane targeting, but did not prevent apoptosis induction by CD95L/CHX. Neither CHX nor CD95L affected protein kinase B phosphorylation, but when added together a marked dephosphorylation of PKB was observed. Conclusion: Sensitization of HSC towards CD95L-induced apoptosis by cycloheximide is accompanied by a JNK-dependent CD95 membrane targeting, which, however, has little impact for the apoptotic response.

Background & Aims:Hepatic stellate cells play an important role in liver fibrogenesis, and hepatic stellate cell death may be involved in the termination of this response. Methods:Molecular mechanisms of hepatic stellate cell killing were studied in hepatic stellate cell/Kupffer cell cocultures. Results:Lipopolysaccharide stimulation of hepatic stellate cell/Kupffer cell cocultures, but not of hepatic stellate cell monocultures, induced profound alterations of hepatic stellate cell morphology and hepatic stellate cell death. Kupffer cell–induced hepatic stellate cell killing required hepatic stellate cell/Kupffer cell contacts and was prevented by dexamethasone, prostaglandin E2, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor 2 antagonists, and down-regulation of receptor-interacting protein, but not by antioxidants, tumor necrosis factor receptor, or CD95 antagonists. Hepatic stellate cell death was characterized by activation of caspases 3, 8, and 9, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling negativity, lack of gross calcium overload, and TRAIL trafficking to the plasma membrane. Inhibition of caspase 9, but not of caspases 3, 8, or 10, prevented hepatic stellate cell death. Lipopolysaccharide induced a dexamethasone- and prostaglandin E2–sensitive expression of TRAIL in Kupffer cells. TRAIL receptors 1 and 2, FLIP (caspase 8–inhibitory protein), and receptor-interacting protein were up-regulated during hepatic stellate cell transformation; however, TRAIL addition did not induce hepatic stellate cell death. Hepatic stellate cell susceptibility toward Kupffer cell–induced death paralleled receptor-interacting protein and TRAIL-receptor expression levels. Conclusions:Activated Kupffer cell can effectively kill hepatic stellate cell by a caspase 9– and receptor-interacting protein–dependent mechanism, possibly involving TRAIL. The data may suggest a novel form of hepatic stellate cell death.

The influence of anisoosmolarity on NF‐κB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF‐κB binding activity whereas the hyperosmotic NF‐κB response was only minor. Hypoosmotic NF‐κB activation was accompanied by degradation of the inhibitory IκB‐α. Protein kinase C, PI3‐kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF‐κB. Hypoosmolarity plus PMA induced, amplified and prolonged IκB‐α degradation and NF‐κB binding activity. Transforming growth factor β‐induced apoptosis was diminished by hypoosmolarity. However, this anti‐apoptotic effect was probably not related to NF‐κB activation.