TNF-α-Induced Sphingosine 1-Phosphate Inhibits Apoptosis Through a Phosphatidylinositol 3-Kinase/Akt Pathway in Human Hepatocytes

Abstract

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.

Expression of the NF-κB Target Gene X-Ray-Inducible Immediate Early Response Factor-1 Short Enhances TNF-α-Induced Hepatocyte Apoptosis by Inhibiting Akt Activation

“…However, its activation was almost abolished when cells were infected with adenovirus expressing the mutant I B (Ad5I B) (25 multiplicity of infection) ( Fig. 1), as previously reported (8). The genes, whose expression levels increased Ͼ2-fold by TNF-␣ (Ad5LacZ ϩ TNF) compared with Ad5LacZ alone, and also were reduced by Ͼ25% by Ad5I B (Ad5I B ϩ TNF) treatment compared with Ad5LacZ ϩ TNF, were regarded as positive and are summarized in Table I.…”

“…Hc cells were cultured in CS-C complete medium supplemented with antibiotics (penicillin, streptomycin) (8). Cells (3 ϫ 10 5 , 5 ϫ 10 5 , and 3 ϫ 10 6 ) were plated on 35-, 60-, and 100-mm dishes, respectively.…”

“…Nuclear extracts were prepared as described previously (8). NF-B-binding consensus single-strand oligonucleotide (5Ј-TAGTTGAGGGGACTTTC CCAGG-3Ј) was first annealed with the complement oligonucleotide (5Ј-TGCCTGGGAAAGTCCCCTCAACTA-3Ј).…”

“…However, hepatocytes are normally resistant to the cytotoxicity of TNF-␣. Administration of TNF-␣ alone does not induce hepatocyte apoptosis in mouse liver in vivo (5-7) or in cultured hepatocytes (8), suggesting that TNF-␣ also activates molecules which protect cells from apoptosis. For example, TNF-␣ transmits antiapoptotic signals via NF-B and phosphatidylinositol 3-kinase (PI3K) 3 /Akt.…”

“…For example, TNF-␣ transmits antiapoptotic signals via NF-B and phosphatidylinositol 3-kinase (PI3K) 3 /Akt. Blockage of these signaling pathways results in sensitization of hepatocytes to apoptosis induced by TNF-␣ (8). NF-B regulates the expression of antiapoptotic gene products (9 -11), such as antiapoptotic members of the Bcl-2 family (12) and the inhibitor of apoptosis proteins (13).…”