A complex growth process such as liver regeneration requires the precise orchestration of a multitude of hormonal, metabolic, cytokine, and growth-factor signals to ensure the precise timing and coordination of DNA replication. Priming and progression of hepatocyte proliferation after PH requires the actions of TNF and IL-6 and growth factors such as TGF-α and HGF (1
). However, the mechanisms that downregulate or “turn off” these signaling pathways is unclear despite their critical role in controlling growth. The studies presented here show that SOCS-3 transcripts and protein are induced during the priming phase of liver regeneration and that this induction is greatly diminished in IL-6 KO mice.
TNF levels increase after PH, activating the transcription factor, NF-κB, which in turn upregulates IL-6 mRNA, resulting in elevated serum levels of IL-6. IL-6 stimulates hepatic STAT-3 by inducing phosphorylation at tyrosine 705, which allows nuclear translocation and DNA binding. STAT-3 mediates transcription of a variety of genes including genes involved in proliferation such as cyclins, cdc25A, and c-myc (reviewed in refs. 28
). STAT-3 also induces SOCS-3 mRNA leading to an increase in SOCS-3 protein levels, which downregulate IL-6–stimulated STAT-3. Both STAT-3 activity and SOCS-3 protein are no longer detectable by 8 hours after PH. Thus in liver regeneration, SOCS-3 participates in a negative-feedback loop turning off IL-6–mediated STAT-3 activation. Inhibition of STAT-3 signaling may ensure the termination of the priming phase of liver regeneration.
SOCS-3 appears to be the major isoform regulating cytokine signaling during liver regeneration. Compared with SOCS-3, which is induced 40- to 60-fold after PH, CIS and SOCS-2 were only weakly induced. Since SOCS-3 induction is severely blunted in IL-6 KO liver after PH, the induction of SOCS-3 after PH is, for the most part, dependent on IL-6. Interestingly, when IL-6 is injected into mice, there are striking differences in the SOCS profiles compared with induction after PH. SOCS-3 mRNA is induced by 1 hour and rapidly declines by 2 hours after IL-6 injection (data not shown). After PH, SOCS-3 mRNA is much more sustained because mRNA is detected between 2 and 8 hours after PH. In addition to SOCS-3, injection of IL-6 induces CIS, SOCS-1, and SOCS-2 mRNA (Figure ) in the liver, an observation reported by other laboratories (20
). In contrast, CIS, SOCS-1, and SOCS-2 are not strongly induced after PH. Besides IL-6, there must be other factors, cytokines, or growth factors, that directly or indirectly influence SOCS-3 induction after PH. Moreover, the lack of induction of the other SOCS isoforms after PH suggests that a single injection of IL-6 does not faithfully reproduce the signaling parameters of PH.
The SOCS-3 induction is not completely abolished in IL-6 KO mice after PH, perhaps uncovering the actions of other cytokines. Clues to the identity of these cytokines or factors maybe revealed in the limited pattern of STAT family members that can bind DNA after PH. Only STAT-3 homodimers are seen after PH (Figure c and ref. 16
). Moreover, both hepatocytes in vivo and in culture respond to IL-6 treatment in a similar manner; that is, only STAT-3 homodimers (SIF A) are found (unpublished observations and ref. 30
). One notable exception is the human hepatoma cell line, HepG2 (Figure c), where SIF A, B (STAT-3:STAT-1), and C (STAT-1:STAT-1) DNA complexes are all detected after IL-6 treatment (30
). Oncostatin M (OSM), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1), members of the IL-6 cytokine family that can induce STAT-3 DNA binding, could be the cytokines that cooperate with IL-6 during regeneration. These cytokines have been implicated in liver functions such as development, hepatocyte differentiation, or inflammation (31
), but have yet to be examined in liver regeneration.
SOCS-3 has not been shown to have catalytic phosphatase activity, yet by 8 hours after PH, STAT-3 is no longer tyrosine phosphorylated (Figure a). Thus two mechanisms, SOCS-3 and a tyrosine phosphatase, may coexist to ensure complete inhibition of cytokine signals mediated by STATs. In the first mechanism, nascent SOCS-3 blocks the activation of STAT-3 by binding to the receptor complex via gp130 at tyrosine 759 (34
) and/or by binding and inhibiting Jak2 activity (36
). Secondly, existing, activated pools of STAT-3 (i.e., tyrosine phosphorylated) may be inactivated by dephosphorylation through the actions of a tyrosine phosphatase. Possible tyrosine phosphatases include an unidentified nuclear tyrosine phosphatase activity that has been reported to inactivate IFN-γ–stimulated STAT-1 (37
) and the SH2-containing phosphatases, SHP-1 and SHP-2, which regulate cytokine signals in other tissues (38
). However, the roles of any of these candidate tyrosine phosphatases in regeneration remain to be examined. Other mechanisms such as PIAS (39
) that downregulate cytokine signaling in other cells may also play a role in inhibiting early cytokine signaling during PH.
The priming phase of hepatocyte replication probably ends at 5–6 hours after PH, perhaps even earlier. Many cytokine signals are downregulated by the end of this time period. SOCS-3 appears to play an important role in inhibiting the IL-6–signaling pathway. While SOCS-3 is induced after PH and the timing of its expression correlates very well with the inactivation of STAT-3 DNA binding, more precise analysis of the role of SOCS-3 awaits the establishment of SOCS-3–transgenic or KO mice. These expression systems require conditional expression of SOCS-3 within the parenchymal cells of the liver since constitutive expression or ablation results in embryonic death (40
). Nevertheless, this analysis of SOCS-3 contributes to our understanding of the termination of signaling pathways involved in liver regeneration.