GH resistance and low serum levels of insulin-like growth factor 1 have been identified in a large percentage of cirrhotic patients (23
), suggesting a link between STAT5 signaling and liver fibrosis and possibly HCC. In this paper, we have demonstrated that TGF-β directly interacts with STAT5 and that elevated levels of TGF-β abrogate GH-induced STAT5 activation. Instead, elevated levels of TGF-β result in GH activation of STAT3, and constitutive STAT3 activity is commonly observed in HCC. Elevated phosphorylation of STAT3 has been associated with epithelial cancers and linked to the induction of STAT3 target genes. In support of a STAT5–TGF-β–STAT3 mechanism, deletion of STAT5 from hepatocytes in mice resulted in elevated levels of TGF-β, an activation of STAT3, and, finally, the development of HCC. We hypothesize that a TGF-β–mediated abrogation of STAT5 signaling results in the activation of STAT3, which is a key step in the development of HCC ().
Most likely, the development of liver cancer in this model is the result of more than one molecular insult. Notably, excessive STAT3 activation upon GH and CCl4
treatment in STAT5-LKO mice could result in unscheduled cell proliferation and transformation. TGF-β binds to the N terminus of STAT5, and loss of STAT5 led to elevated TGF-β levels as a result of increased stability. The absence of the STAT5 N terminus in STAT5-ΔN mice did not abrogate the development of liver fibrosis, but no tumors were observed in these mice. This suggests that an N-terminally truncated STAT5 still contains tumor suppressor functions similar to that seen with wild-type STAT5. Several studies that compared STAT5-ΔN mice and mice completely missing STAT5A/B established functional activity of the N-terminally truncated STAT5. For example, although the complete loss of STAT5 results in perinatal lethality, STAT5-ΔN mice are viable (33
). Similarly, the complete loss of STAT5 causes more severe hematological defects (34
). A recent study demonstrated that the N-terminal domain plays an important role in maintaining normal balance of lymphoid and myeloid cells (36
). Although it is clear that loss of STAT5 N terminus, which is required for the tetramerization of STAT5, does not result in increased liver cancer induced by CCl4
, its contribution to oncogenesis might be cell specific. Moriggl et al. (37
) established that loss of the N-terminal region protected the development of certain leukemia. At this point, the cell specificity of STAT5 in normal development and oncogenesis is poorly understood.
TGF-β and STAT3 are not only critical in the promotion of tumor progression and survival but they also contribute to tumor-immune escape (38
). Notably, increased TGF-β secretion might lead to the expansion of CD4+
regulatory T (T reg) cells that would impact liver fibrosis and tumor development (40
). Another possibility for the promotion of liver fibrosis is that the deletion of STAT5 might have an effect on HSC or KC and increase their sensitivity to TGF-β. However, the CD4/CD8 ratio and TH1- and TH2-cytokine production were similar in control and mutant mice (Fig. S8, A–C
), and no selective expansion of T reg cells was observed after CCl4
treatment (Fig. S8, D and E). Moreover, the sensitivity of HSC to TGF-β in isolated HSC was identical in both mice (Fig. S9
). These results contributed to a nonbiased analysis of the role of STAT5 in liver fibrosis and cancer development.
In the absence of inflammation, liver TGF-β is secreted from HSC and KC but not from hepatocytes. However, upon inflammation or injury hepatocytes gradually become the major source of TGF-β (42
). As hepatic fibrosis develops, TGF-β levels increase, followed by the accumulation of extracellular matrix (4
). Collectively, hepatocytes secrete an excess of TGF-β in the late stage of liver fibrosis when most cancer develops. It has been shown that TGF-β1 induces an epithelial-to-mesenchymal transition in mature hepatocytes, resulting in type I collagen synthesis (46
). Because embryonic fibroblasts secrete TGF-β, MEFs are reminiscent of myofibroblasts, which are activated in liver fibrosis state. TGF-β levels were highly elevated in STAT5-null MEFs and reintroduction of STA5 resulted in the suppression of TGF-β. Based on our studies, we propose that binding of STAT5 to TGF-β reduces its half-life. STAT5 has been reported to interact with other molecules, such as the glucocorticoid receptor (GR) and HNF (hepatocytes nuclear factor) 4α (48
). Although the GR can act as a transcriptional coactivator of STAT5 and enhances STAT5-dependent transcription, the GR–STAT5 complex diminishes the glucocorticoid response of promoters harboring glucocorticoid response elements. As for HNF-4α, it strongly inhibits GH-induced STAT5B transcriptional activity. Although STAT5 decreases TGF-β stability, resulting in a reduction of TGF-β, excess levels of TGF-β inhibit GH-induced activation of STAT5. In this context, GH preferentially activates STAT3, which in turn activates genes linked to cell proliferation and survival. Thus, STAT5 transcriptional functionality can be modulated by activating and suppressing cofactors (50
The unscheduled ability of STAT3 to be activated by cytokines that normally only activate STAT5 is no artifact of the STAT5-KO mouse model. Recently, several inactivating STAT5B mutations have been identified in humans (51
) and linked to dwarfism and immunological disorders. Stimulation of primary cells from these patients with GH and prolactin leads to an unscheduled activation of STAT3 (52
), and it is conceivable that some of the immunological disorders observed in these patients can be linked to the aberrant activation of STAT3 rather than the loss of STAT5B. With respect to liver fibrosis and HCC, we propose that TGF-β–mediated down-regulation of STAT5 levels in hepatocytes leads to an aberrant activation of STAT3.