The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors.

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We previously observed that Gadd45␤/MyD118, a member of the Gadd45 family of inducible factors, showed the strongest immediate-early induction common to two distinctive proliferation responses of the liver: (1) regeneration induced by surgical partial hepatectomy and (2) hyperplasia induced by the primary mitogen TCPOBOP, a ligand of the constitutive androstane receptor (CAR). Gadd45␤ is known to be stimulated by nuclear factor (NF) B, which is activated by tumor necrosis factor alpha (TNF␣) in the early response to partial hepatectomy. We therefore investigated whether TNF␣ and NF B also stimulated Gadd45␤ as part of the response to CAR ligands, or whether activation occurred by an alternative pathway. TCPOBOP effects were characterized in three mouse genotypes: wild-type, TNFR1 ؊/؊ , and TNFR1 ؊/؊ TNFR2 ؊/؊ . The results showed that TCPOBOP did not activate NF B in any of the mice, but a strong induction of Gadd45␤ messenger RNA was observed in all three genotypes, where TCPOBOP also induced CyP2b10, a classical target gene of activated CAR, and cyclin D1, a proliferation linked gene. Thus, the absence of TNFR signaling and induction of NF B did not impair CAR-mediated gene induction. Moreover, hepatocyte proliferation was strongly induced, and at significantly higher levels than wild type, in both TNFR1 I n recent years, the tools of molecular biology have revealed important mechanisms controlling liver regeneration, including critical growth factors, transcription factors, and signal transduction regulators. [1][2][3][4] Within minutes after partial hepatectomy (PH), hepatocytes in the remnant liver undergo a transition from the quiescent G0 to the G1 phase of an active cell cycle. Although the precise mechanisms responsible for triggering this transition are not known, enhanced expression of immediate-early genes, occurring 15 minutes to 2 hours after PH, is believed to be critical. 5 Immediate-early gene induction does not require protein synthesis and is activated by transcription factors that preexist in a latent form. In particular, increased binding of nuclear factor (NF) B, AP1, and C/EBP occurs within minutes after PH, 6-8 whereas STAT3 activates shortly thereafter. Activation of AP1 and NF B, as well as DNA synthesis, can be inhibited by pretreatment of the animals with antibodies against tumor necrosis factor alpha (TNF␣), 9 suggesting that this cytokine plays a central role in the initiation of liver regeneration, by activating transcription factors.

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Partial hepatectomy (PH) and some tumor-promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate-early gene expression changes after PH with those induced by 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP), a tumor-promoting liver mitogen. PH activates nuclear factor B (NF-B) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up-regulation or down-regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty-three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest downregulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components. (HEPATOLOGY 2003;38:314-325.)