Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals.

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Tumor necrosis factor (TNF)-␣ signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the "ceramide/ S1P rheostat") may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-␣-induced apoptosis of primary hepatocytes. Endogenous C 16 -ceramide was elevated during TNF-␣-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-␣-induced apoptosis and C 16 -ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-␣-induced increase of C 16 -ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with D-galactosamine plus TNF-␣. This protective effect was abrogated by the sphingosine kinase inhibitor N,Ndemethylsphingosine, suggesting that the survival effect of NCDase is due to not only C 16 -ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C 16 -ceramide contributed to TNF-␣-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C 16 -ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-␣.

Human hepatocytes usually are resistant to TNF-α cytotoxicity. In mouse or rat hepatocytes, repression of NF-κB activation is sufficient to induce TNF-α-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IκBα (Ad5IκB), which almost completely blocks NF-κB activation, >80% of the cells survived 24 h after TNF-α stimulation. Here, we report that TNF-α activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-α in Ad5IκB-infected Huh-7 and Hc cells. TNF-α-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-α-induced apoptosis. Although Akt has been reported to activate NF-κB, DMS and LY 294002 failed to prevent TNF-α-induced NF-κB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-κB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-κB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-α and probably FasL.

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