The p105/50 NF-kappaB pathway is essential for Mallory body formation.

To determine if nuclear factor-kappaB (NF-kB) plays a role in Mallory body (MB) formation, quantitative real-time RT-PCR assay was used to measure liver NF-kappaB1/p105 mRNA levels in 4 different groups of mice. Group 1: mice given IP saline for 15 weeks; group 2: mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks when MBs were formed; group3: mice fed DDC 10 weeks, then withdrawn 5 weeks when MBs disappeared; group 4: mice fed DDC 10 weeks, withdrawn 4 weeks, then fed DDC+chlormethiazole (CMZ) for 1 week when MBs again formed. The mRNA for p105 NF-kappaB expression was significantly increased in the livers of mice treated with DDC (group 2) and DDC+CMZ (group 4) compared with the control livers (group 1) as well as the drug-withdrawal livers (group 3). Primary cultures of hepatocytes from drug-primed mice (the group 4 mice were withdrawn for another 4 weeks when the MBs had disappeared) were studied. The hepatocytes from drug-primed mice were MB free when isolated and used for primary culture. MBs began to form spontaneously within their cytoplasm after 2-3 days of culture. The NF-kappaB inhibitor (NF-kappaBi), a cell-permeable quinazoline compound that acts as a potent inhibitor of NF-kappaB transcriptional activation, was added to the medium 3 h after planting the cultures of liver cells. No MBs formed in the cells treated with 10 microM, 1 microM, and 0.1 microM NF-kappaBi for 6 days. MBs still formed in the cells treated with 10 nM NF-kappaBi for 6 days. Both DDC-primed and normal control liver cells began to enlarge and elongate after a few hours of culture. In contrast, the cells treated with NF-kappaBi stayed polyhedral in shape just as they appeared prior to culturing. The level of NF-kappaB1/p105 mRNA significantly increased in DDC-primed hepatocytes after 24 h of culture and in normal control hepatocytes after 48 h of culture. In DDC-primed hepatocytes, NF-kappaBi 0.1 muM treatment for 6 days significantly decreased mRNA expression of Src, p105/NF-kappaB1, ERK1, MEKK1, and JNK1/2. In normal control liver cells, NF-kappaBi treatment decreased mRNA expression of Src and JNK1 and stimulated the mRNA expression of p105/NF-kappaB1 and Junk2. NF-kappaBi treatment significantly decreased the total ERK1/2 protein and further decreased the phosphorylated (activated) form of ERK1/2 in the cultured hepatocytes. The results indicate that the p105 NF-kappaB pathway which putatively regulates ERK at both the transcriptional and post-translational levels regulates MB formation by way of changes in gene expression.

The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study.

It is still unclear as to how hepatocytes perceive external factors and transduce the signals which initiate MB formation. To investigate this phenomenon, the model of MB formation in liver in vivo and in primary culture of hepatocytes derived from drug-primed mice was used. Control mice were fed the control diet (group 1). MBs were induced in the livers of mice fed diethyl-1, 4-dihydro-2, 4, 6-trimethyl-3, 5-pyridinedicarboxylate (DDC) for 10 weeks (group 2). The induced MBs completely disappeared after the withdrawal of DDC for 4 weeks (group 3). Newly formed MBs were numerous after DDC was refed for 1 week (group 4). Relative mRNA abundance was determined by quantitative real-time RT-PCR in the liver from the mice. The expression of integrin alpha(6) and beta(2) was significantly increased in the livers of DDC-treated (group 2) and drug refed mice (group 4), when compared with the livers from controls (group 1) and DDC-withdrawn (group 3) mice. The increased mRNA of these two integrin genes was associated with the increased expression of laminin (a ligand for integrin alpha(6)beta(1) and alpha(6)beta(4)), Icam1 (a ligand of alphaLbeta2), Src, MEKK1, and ERK1. Primary cultures of isolated DDC-primed hepatocytes (group 4 mice were withdrawn from DDC-CMZ for 4-6 weeks) produced significantly more MBs on laminin-coated coverslips compared with plastic uncoated, fibronectin-, collagen-, or fibrinogen-coated coverslips. U0126, an inhibitor of MEK1 protein, significantly reduced the phosphorylated forms of ERK1/2 and MB formation in vitro. In conclusion, the current study revealed an association between MB formation and integrin-mediated signaling in vivo. The data indicate that laminin-integrin signaling which activates ERK, triggered MB formation in vitro, and an inhibitor of the signaling cascade reduced MB formation.