Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and a-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/ FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGFa was blocked by antioxidants, such as da-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGFa and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGFa. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the a-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation. (J. Clin. Invest. 1995. 96:2461-2468

Muscle wasting is a critical feature of patients afflicted by AIDS or cancer. In a murine model of muscle wasting, tumor necrosis factor alpha (TNF alpha) induces oxidative stress and nitric oxide synthase (NOS) in skeletal muscle, leading to decreased myosin creatinine phosphokinase (MCK) expression and binding activities. The impaired MCK‐E box binding activities resulted from abnormal myogenin‐Jun‐D complexes, and were normalized by the addition of Jun‐D, dithiothreitol or Ref‐1, a nuclear redox protein. Treatment of skeletal muscle cells with a phorbol ester, a superoxide‐generating system, an NO donor or a Jun‐D antisense oligonucleotide decreased Jun‐D activity and transcription from the MCK‐E box, which were prevented by antioxidants, a scavenger of reducing equivalents, a NOS inhibitor and/or overexpression of Jun‐D. The decreased body weight, muscle wasting and skeletal muscle molecular abnormalities of cachexia were prevented by treatment of TNF alpha mice with the antioxidants D‐alpha‐tocopherol of BW755c, or the NOS inhibitor nitro‐L‐arginine.

Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl(4) induced activation of RSK, phosphorylation of C/EBPbeta on Thr(217), and proliferation of stellate cells in normal mice, but caused apoptosis of these cells in C/EBPbeta-/- or C/EBPbeta-Ala(217) (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPbeta-PThr(217) and the phosphorylation mimic C/EBPbeta-Glu(217), but not C/EBPbeta-Ala(217), were associated with procaspases 1 and 8 in vivo and in vitro and inhibited their activation. Our data suggest that C/EBPbeta phosphorylation on Thr(217) creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T(217)VD) that is mimicked by C/EBPbeta-Glu(217) (KE(217)VD). C/EBPbeta-/- and C/EBPbeta-Ala(217) stellate cells were rescued from apoptosis by the cell permeant KE(217)VD tetrapeptide or C/EBPbeta-Glu(217).

We report that TGF alpha induces activation of the p90 ribosomal S kinase (RSK), which results in the phosphorylation of rat C/EBP beta on Ser-105 and of mouse C/EBP beta on Thr-217 and concomitantly stimulates proliferation in differentiated hepatocytes. Moreover, C/EBP beta-/- mouse hepatocytes respond to TGF alpha when wild-type C/EBP beta is reexpressed, whereas they remain refractory to the growth effect of TGF alpha when expressing phosphoacceptor mutants rat C/EBP beta Ala-105 or mouse C/EBP beta Ala-217. In contrast, C/EBP beta-/- hepatocytes expressing the phosphorylation mimic mutants, rat C/EBP beta Asp-105 or mouse C/EBP beta Glu-217, exhibited marked proliferation in the absence of TGF alpha. Thus, a site-specific phosphorylation of the transcription factor C/EBP beta is critical for hepatocyte proliferation induced by TGF alpha and other stimuli that activate RSK.