Increased activation of JNK in the liver of p38α^LPC-KO mice by LPS injection. (A) Phosphorylation of JNK was assessed in liver extracts from wild-type (WT) and p38α^LPC-KO mice by immunoblot analysis using phospho-JNK-specific antibodies (upper panel). JNK immunoblot acts as a loading control. (B) Immunoblot analysis of phosphorylation of JNK in NEMO^LPC-KO and control (WT) mice. (C) Immunoblot analysis with antibodies recognizing phosphorylated c-Jun (upper panel), total c-Jun (middle panel) and tubulin (lower panel) as a loading control. (D) The levels of c-FLIP(L) in WT, p38α^LPC-KO and NEMO^LPC-KO mice were analysed by immunoblot at the indicated time points after LPS injection. JNK, c-Jun amino-terminal kinase; LPS, lipopolysaccharide.

p38α collaborates with IKK2 to protect the liver from LPS-induced toxicity. (A) Immunoblot analysis for the expression of IKK2 and p38α in the extracts of liver protein from wild-type (WT), p38α^LPC-KO and p38α/IKK2^LPC-KO mice. (B) Levels of free circulating ALT were measured in IKK2^LPC-KO, p38α/IKK2^LPC-KO and control (WT) mice before and 10 h after LPS injection. Error bars denote s.e.m. ^*Statistical significance by Student's t-test with P<0.05 relative to control (n=4). Mean values are depicted above each column. (C) Detection of apoptotic cells by TUNEL assay in liver sections from WT, IKK2^LPC-KO and p38α/IKK2^LPC-KO mice 10 h after LPS injection. (D) Immunoblot analysis of caspase 3 activation using antibodies that specifically detect total caspase 3 (top panel) or the cleaved form of caspase 3 (middle panel) in liver extracts from mice with the indicated genotypes 10 h after LPS injection. NEMO^LPC-KO mice were used as a positive control. Tubulin acts as a loading control. (E) The levels of c-FLIP(L) were measured by immunoblot analysis in livers from WT, p38α^LPC-KO and p38α/IKK2^LPC-KO mice at the indicated time points after LPS injection. Each lane represents an individual mouse. ALT, alanine aminotransferase; DAPI, 4,6-diamidino-2-phenylindole; IKK, IκB kinase; LPS, lipopolysaccharide; TUNEL, TdT-mediated dUTP nick end labelling.

p38α^LPC-KO mice are not sensitive to LPS-induced liver failure. (A) Schematic description of the targeting strategy for the generation of mice with loxP-flanked p38α alleles. Filled boxes indicate the loxP-flanked exons 2 and 3 (E2, E3), which include the ATP-binding site of the kinase domain. B, BamHI; H, HindIII. Black arrowheads indicate loxP sites; white arrows indicate FLP recombinase target (Frt) sites. (B) Immunoblot analysis of expression of p38α in liver extracts from wild-type (WT) and p38α^LPC-KO mice. (C–E) Assessment of liver damage in p38α^LPC-KO and control mice after LPS injection. (C) Levels of free circulating alanine aminotransferases (ALTs) were measured in the serum of p38α^LPC-KO and control mice at the indicated time points after LPS injection. Error bars denote s.e.m. (n=4). (D) Detection of apoptotic cells by TUNEL assay in livers from WT, p38α^LPC-KO and NEMO^LPC-KO (used as a positive control) mice 10 h after LPS injection. (E) Immunoblot analysis of caspase 3 activation using an antibody that specifically detects the cleaved form. DAPI, 4,6-diamidino-2-phenylindole; LPS, lipopolysaccharide; TUNEL, TdT-mediated dUTP nick end labelling.

Hyperphosphorylation of JNK in the liver of LPS-injected p38α^LPC-KO correlates with increased activation of MKK4. (A) Wild-type (WT; lanes 1–3), p38α^LPC-KO (lanes 4–6) and p38α^LPC-KO mice that had been pretreated with the antioxidant compound BHA (lanes 7 and 8) were injected with LPS and killed at the indicated time points. Immunoblot analysis was carried out on extracts of liver protein using antibodies against phosphorylated JNK or total JNK proteins. (B) Immunoblot analysis of the phosphorylation status of the JNK-activating kinases MKK4 and MKK7 in the liver extracts from WT and p38α^LPC-KO mice at the indicated time points after LPS injection. Antibodies recognizing phosphorylated or total MKK7 or MKK4 were used. Tubulin acts as a loading control. n.s. indicates a nonspecific band. (C) Immunoblot analysis using an antibody detecting the phosphorylated forms of MKK3 and MKK6 (upper panel). Immunoblots with antibodies against total MKK3 and tubulin (lower panels) act as a loading control. BHA, butylated hydroxyanisole; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MKK, mitogen-activated protein kinase kinase.

p38α collaborates with IKK2 to prevent liver failure in response to in vivo LPS/TNF challenge. TNF binding to TNFRI induces the activation of NF-κB and MAPK pathways, but it can also induce cell death through the activation of caspase 8. Activation of NF-κB protects cells from TNF-induced cell death by inducing the expression of anti-apoptotic proteins such as c-FLIP. Activation of JNK induces the E3 ubiquitin ligase ITCH to ubiquitinate c-FLIP leading to its degradation. Lack of p38α in hepatocytes leads to hyperactivation of MKK3/6, MKK4 and JNK on in vivo LPS challenge. The increased sustained activation of JNK is not sufficient to induce cell death in the p38α-deficient liver. When p38α ablation is combined with moderate inhibition of NF-κB, achieved by hepatocyte-restricted IKK2 ablation, in vivo LPS challenge results in increased degradation of c-FLIP and liver damage through caspase 8-mediated hepatocyte apoptosis. IKK, IκB kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; TNF, tumour necrosis factor.