Tumor Necrosis Factor Induces DNA Replication in Hepatic Cells through Nuclear Factor k B Activation 1

Tumor necrosis factor (TNF) signaling through TNF receptor 1 (TNFR1) with downstream participation of nuclear factor kB (NFkB), interleukin 6 (IL-6), and signal transducers and activators of transcription 3 (STAT3) is required for initiation of liver regeneration. It is not known whether the proliferative effect of TNF on hepatocytes is direct or requires the participation of Kupffer cells, the liver resident macrophages. Moreover, it has not been determined whether NFkB activation is an essential step in TNF-induced proliferation. To answer these questions, we conducted studies in LE6 cells, a rat liver epithelial cell line with hepatocyte progenitor capacity. We report that TNF induces DNA replication in growth-arrested LE6 cells and that its effect involves the activation of NFkB and STAT3 and an increase in c-myc and IL-6 mRNAs. All of these effects, which mimic the events that initiate liver regeneration in vivo, are blocked if NFkB activation is inhibited by expression of a dominant-inhibitor IkBa mutant (DN-IkBa). Although NFkB blockage by DN-IkBa causes caspase activation and massive death of cells stimulated by TNF, inhibition of NFkB and STAT3 binding by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone results in G0-G1 cell cycle arrest without death. We conclude that NFkB is an essential component of the TNF proliferative pathway and that TNF-induced changes in IL-6 mRNA, STAT3, and c-myc mRNA are dependent on NFkB activation. Blockage of NFkB inhibits TNF-induced proliferation but does not necessarily cause cell death. Introduction TNF is a pleiotropic cytokine that participates in a wide range of biological activities, including inflammation, growth, differentiation, and apoptosis. TNF is a key molecule in the cytokine network, capable of regulating the expression of other cytokines, such as IL-6. It is produced as a membranebound protein and is released in the serum after being processed into the mature soluble form (1). The cytokine exerts its effects by binding to two distinct receptors, TNFR1 and TNFR2, which are ubiquitously expressed (2). Signaling through TNFR1 is responsible for most of the TNF effects, including activation of the transcription factor NFkB and apoptosis (3). TNFR2 appears to be the main receptor for membrane-bound TNF and for TNF effects on thymocyte proliferation. TNF is a mediator of the hepatic acute phase response to inflammation (1). However, experiments in which hepatocyte DNA synthesis after PH was inhibited in livers of rats pretreated with anti-TNF antibodies indicated that TNF may also have a role in hepatocyte proliferation (4). Using mice deficient in TNFR1 or TNFR2, we found that TNF signaling through TNFR1, but not TNFR2, is required for the initiation of liver regeneration after PH (5, 6). The drastically reduced DNA synthesis in the liver remnant of TNFR1 knockout mice was associated with low levels of IL-6, along with decreased activation of the transcription factors NFkB, STAT3, and AP1. Activation of these factors after PH primes quiescent hepatocytes to make them competent to proliferate. In TNFR1 knockout mice, normal DNA synthesis and STAT3 activation but not NFkB binding were restored by a single injection of IL-6 shortly before PH. Cressman et al. (7) showed that IL-6 knockout mice have decreased activation of STAT3 and AP1, diminished cyclin D1 expression, impaired DNA synthesis, and high mortality after PH. Injection of IL-6 corrected all defects and restored the normal proliferative response in these animals (7). NFkB activation after PH was normal in IL-6 knockout mice and did not change after IL-6 injection. Together, the results from studies of IL-6 and TNFR1 knockout mice suggest that hepatocyte proliferation during liver regeneration can be triggered by a signal transduction pathway that involves TNFR1, NFkB, IL-6, and STAT3. Although these and other reports demonstrated that TNF causes NFkB and STAT3 activation in the liver, it is not known whether NFkB activation is required for DNA replication and to what extent IL-6 production and STAT3 binding in the liver relate to NFkB activity. Moreover, it has been suggested that the TNF effect on hepatocyte proliferation might be indirect, requiring the involvement of Kupffer cells. TNF could induce hepatocyte replication by a two-step process, Received 6/15/99; revised 10/11/99; accepted 11/1/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grants CA23226 and CA74131 from the NIH. M. C. was supported by Training Grants CA09437 and GMO7270. 2 To whom requests for reprints should be addressed, at Department of Pathology, Box 357470, University of Washington School of Medicine, C-516 Health Sciences Building, Seattle WA 98195-7470. Phone: (206) 685-1221; Fax: (206) 543-3644; E-mail: nfausto@u.washington. edu. 3 The abbreviations used are: TNF, tumor necrosis factor; TNFR, TNF receptor; IL, interleukin; NF, nuclear factor; PH, partial hepatectomy; STAT, signal transducers and activators of transcription; AP, activating protein; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; BrdUrd, bromodeoxyuridine; EMSA, electrophoretic mobility shift assay; CHX, cycloheximide; wt, wild type; OSM, oncostatin M. 819 Vol. 10, 819–828, December 1999 Cell Growth & Differentiation