An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-α

Tumor necrosis factor-α (TNF) can stimulate multiple disparate hepatocyte responses—proliferation, survival, or apoptosis—depending on the cellular context. TNF binds and activates the receptor TNFR, leading to downstream activation of the c-Jun N-terminal kinase (JNK), inhibitor of nuclear factor-κB (IκB) kinase (IKK)–nuclear factor (NF)-κB, and p38 signaling pathways and caspase cascade.1 The modulation of these and other intracellular signaling pathways by concomitant synergistic and antagonistic cytokine stimuli, viruses, and/or pharmacological treatments determine specific cell responses to TNF.2–4 In the partial hepatectomy (PHx) model of liver regeneration, normally quiescent hepatocytes are stimulated to proliferate in a process regulated by multiple redundant signaling pathways and molecules.5 Following PHx, Kupffer cells are activated and secrete TNF and interleukin (IL)-6. These cytokines stimulate the transcription of a set of “immediate early” genes and a G0–G1 cell cycle progression in hepatocytes.6,7 TNF signaling primes hepatocytes for DNA replication through subsequent stimulation by hepatocyte growth factor and epidermal growth factor receptor (EGFR) ligands.5,7–9 In primary hepatocyte cultures, TNF not only potentiates growth factor-stimulated proliferation, but acts as a mitogen itself6,10 through the induced release of autocrine transforming growth factor-α (TGF-α) and its activation of serine-threonine protein kinase B (PKB)/Akt and extracellular signal-regulated kinase (ERK).11,12 The IL-1 receptor (IL-1R) agonists IL-1α and IL-1β antagonize hepatocyte proliferation when produced by nonparenchymal cells in vivo during liver regeneration and when added exogenously to mitogenic factors in vitro13,14 Hepatocytes are resistant to apoptosis stimulated by TNF alone as it activates both proapoptotic and anti-apoptotic signaling pathways.1,4 Consequently, pharmacologic or genetic interference with antiapoptotic signaling is commonly used to examine TNF-induced hepatocyte apoptosis.4 In diseased and/or virus-infected hepatocytes, TNF signaling contributes to apoptotic and necrotic cell death.4,15 Recently, we have shown that infection with a replication-deficient adenoviral vector (Adv) potently sensitizes human epithelial cell lines, including the C3A hepatocarcinoma cell line, to TNF-induced apoptosis via both proapoptotic and antiapoptotic signaling pathways.3 Adenoviral gene therapy vectors targeting the liver and other organs are often compromised due to hepatocyte death induced by both the viral vector itself and cytokines of the innate immune response such as TNF and IL-1β.16 Therefore, Adv infection might provide a physiologically relevant environment to potentiate TNF-induced apoptosis in primary hepatocytes and could lead to insights in liver adenoviral gene therapy. Hepatocyte death responses to TNF and other inflammatory cytokines can be antagonized by many of the same growth factors that stimulate hepatocyte proliferation8,17 or by naturally occurring inhibitors of cytokine signaling such as IL-1 receptor antagonist (IL-1ra).18 Whereas many of the factors, such as TNF, that affect hepatocytes during injury or stress arise primarily from exogenous sources, hepatocytes themselves secrete growth factors and cytokines that act in autocrine fashion to enhance or oppose exogenous stimuli.8,9,11,12,17,19 Recently, we have demonstrated that the response of human epithelial cell lines to TNF involves release of TGF-α, IL-1α, and IL-1ra, which provide conflicting and interlinked autocrine feedback signals governing apoptotic responses to TNF.2,20 Hepatocytes express TGF-α, IL-1α, IL-1β, and IL-1ra and their receptors but it is unknown whether these ligands operate via interlinked autocrine circuits to modulate hepatocyte proliferation and apoptosis responses to TNF.9,12,21,22 Here we show that rat hepatocyte proliferation and apoptosis responses to TNF are both mediated by an inducible, coupled, and self-antagonizing TGF-α–IL-1α/β–IL-1ra autocrine cascade. The net effect of this coupled autocrine cascade is pro-proliferative as induced by TNF alone but proapoptotic when induced by TNF in Adv-infected hepatocytes. Moreover, elucidation of this self-antagonizing autocrine cascade is a useful paradigm that helps rationalize the diverse landscape of hepatocyte phenotypic responses to TNF and TGF-α costimulation and their induction of autocrine IL-1α/β signaling.