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Proteolysis-inducing factor regulates hepatic gene expression via the transcription factors NF-B and STAT3

Molecular Immunology Group, Department of Clinical and Surgical Sciences, Edinburgh University, U.K.; and
^* Cardiovascular and Gastrointestinal Discovery Department, Astrazeneca, Macclesfield, U.K.

²Correspondence: Lister Research Laboratories, RIE, Lauriston Place, Edinburgh EH3 9YW, United Kingdom. twatchorn@ed.ac.uk or J.A.Ross@ed.ac.uk

SPECIFIC AIMS

A novel molecule, proteolysis-inducing factor (PIF) of molecular weight 24,000, appears to fulfill the function of triggering muscle proteolysis during the process of cancer cachexia. PIF has been purified from a cachexia-inducing mouse tumor and from the urine of patients with pancreatic carcinoma and weight-loss. PIF is capable of inducing muscle protein degradation in isolated gastrocnemius muscle preparations and of inducing weight loss in vivo. It is unlikely that PIF is expressed uniquely by tumor cells, but its normal, constitutive, role is still unclear. However, comprehensive tissue screening has demonstrated that only skeletal muscle and liver exhibit substantial binding of PIF. To further ascertain the function and signaling capacity of PIF, we have investigated the biological effect of this molecule on hepatic gene expression in primary cultures of human hepatocytes and on the human cell line HepG2. We have determined the effect of PIF on both the NF-B and STAT3 transcriptional pathways.

PRINCIPAL FINDINGS

1. PIF induces NF-B activation in primary hepatocytes and the HepG2 cell line
Primary hepatocytes were seeded at subconfluent levels and HepG2 cells were grown to subconfluency. Both cell types were treated with PIF, tumor necrosis factor (TNF-) or lipopolysaccharide (LPS) in medium containing 10% serum. Although it has been postulated that PIF binds to albumin, parallel experiments have been carried out in which cells were treated in the absence of fetal calf serum and no differences were observed. Cells were treated for 15, 30, and 60 min and nuclear extracts were prepared to determine whether NF-B activation occurred. Both primary hepatocytes (Fig. 1 ) and HepG2 cells show PIF-induced NF-B activation at 30–60 min, whereas TNF- activation of NF-B occurred at 15–30 min.

View larger version (57K): [in this window] [in a new window]   Figure 1. Electrophoretic mobility shift assay (EMSA) of NF-B in primary hepatocytes at 15, 30, and 60 min. Lane 1: control; lane 2: TNF-; lane 3: PIF 10 ng/ml; lane 4: PIF 100 ng/ml.

2. NF-B activation by PIF results in increased IL-8 and IL-6 production and ICAM-1 expression in hepatocytes
The effect of PIF on the production of the NF-B regulated cytokines, interleuken (IL)-8, and IL-6 was investigated in both primary hepatocytes and HepG2 cells following incubation for 24 h. PIF significantly induces the production of IL-8 in both cell types, but IL-6 in primary hepatocytes only. Messenger RNA (mRNA) for both IL-8 and IL-6 was also detected in the hepatocytes, whereas IL-6 message was absent in HepG2 cells, which suggests that these cells may have lost the ability to produce IL-6. Because TNF- did not appear to have an effect on IL-6 induction in primary hepatocytes, LPS was used as a positive control.

We investigated the effect of PIF on ICAM-1 induction in both primary hepatocytes and HepG2 cells following incubation for 24 h. PIF (100 ng/ml) significantly increases ICAM-1 expression to a level comparable with TNF- in primary hepatocytes. However, no change in ICAM-1 expression was observed in HepG2 cells.

3. PIF induces STAT3 activation in primary hepatocytes
We used the nuclear extracts prepared for NF-B to determine the effect of PIF on STAT3 activation. Figure 2 shows the effect of both IL-6 and PIF on STAT3 activation in primary hepatocytes. STAT3 is activated by PIF at 15–30 min, whereas IL-6 does not induce STAT3 until 30 min. STAT3 activation was not observed in HepG2 cells.

View larger version (86K): [in this window] [in a new window]   Figure 2. Electrophoretic mobility shift assay (EMSA) of STAT3 in primary hepatocytes at 15, 30, and 60 min. Lane 1: control; lane 2: IL-6; lane 3: PIF 10 ng/ml; lane 4: PIF 100 ng/ml.

4. C-reactive protein (CRP) production is increased and transferrin production decreased by PIF in primary hepatocytes
We examined the effect of PIF on hepatic gene expression by using the acute phase protein CRP and the normal export protein transferring. After incubation for 48 h, CRP production was increased significantly by PIF acting on primary hepatocytes, although this increase was less than that observed with IL-6. In addition, PIF significantly decreased transferrin levels in primary hepatocytes, although again, the change was not as great as that observed with IL-6. No CRP or transferrin production was observed from HepG2 cells.

CONCLUSIONS

The biological functions of PIF, beyond the induction of muscle degradation, were unknown. This study investigated the effect of PIF on hepatic gene expression via the NF-B and STAT3 pathways. We demonstrate that PIF can induce NF-B activation in primary hepatocyte cultures and in the hepatoma cell line HepG2. NF-B activation is central to the regulation of many genes, including those involved in growth control and inflammatory processes. We have used IL-8, IL-6, and ICAM-1 as NF-B inducible genes and have demonstrated that NF-B activation results in an increase in both IL-8 and IL-6 production and an increase in ICAM 1 expression in hepatocytes. These results suggest that PIF may play a role in hepatic gene regulation.

PIF has no homology with known cytokine families and is distinguished from other cytokines by the ability to accelerate the breakdown of skeletal muscle both in vitro and in vivo. In addition to the presence of PIF, proinflammatory cytokines such as IL-6, IL-8 and TNF- also increases during the process of cachexia. In this study, we have demonstrated that PIF can increase proinflammatory cytokine release via the transcription factor NF-B. This ability may contribute to the role of PIF in cachexia via its ability to induce the production of proinflammatory cytokines, and ample evidence supports the hypothesis that cachexia is, to some extent, a chronic inflammatory process.

Furthermore, we have also shown that PIF can induce activation of the STAT3 pathway, which is involved in the induction of the acute-phase protein response. STAT3, a member of the STAT family, is activated by a variety of cytokines, including the IL-6 family, leptin, and epidermal growth factor. We have also demonstrated that PIF can increase CRP production and decrease transferrin production, although these changes are not as great as those seen with IL-6 alone, a known inducer of the acute phase proteins via the STAT3 pathway.

Although NF-B and STAT3 are distinct transcription pathways, ligand binding to the appropriate receptors for activation of each transcription factor can also result in activation of the mitogen activated protein kinase pathway, which results in activation of the transcription factor NF-IL-6, and hence it is likely that PIF would also activate NF-IL-6. Thus, tumor-derived PIF may induce a coordinated response involving NF-B, STAT3, and NF-IL-6. A combined effect of the three transcription factors on gene regulation during cancer cachexia would result in increased proinflammatory cytokine and acute-phase protein production.

However, because it is unlikely that PIF is a novel protein produced only by certain tumors, these results may also give some insight into the normal, constitutive, function of PIF. The presence of PIF in pancreatic cancer patients may be explained by the possibility that certain tumors either increase the production of PIF to measurable levels during cancer cachexia or that the tumor alters the protein in some way, possibly through glycosylation. The more likely explanation is that PIF might be inappropriately expressed by some tumor cells. Preliminary studies in mice (unpublished data) demonstrate that PIF is expressed during the embryonic period E8 to E9 and that this expression peaks during E8.5. E8.5 is a crucial stage in the patterning and eventual development of skeletal muscle in the mouse and is associated with myogenin expression. Both myogenin and PIF levels fall to undetectable levels after E9.5 but, unlike myogenin whose expression is skeletal muscle specific, the expression of PIF is more diffuse and is present in skeletal muscle and other tissues, including liver. Thus it is possible that the normal role of PIF may be in the sculpting both skeletal muscle and, possibly, other tissues during vital stages of embryological development.

In addition to its well-established roles in the activation of transcription of genes, NF-B also functions in promoting cell growth. NF-B has been shown to be required for growth of tissues, particularly in the developmental phases; its ability to regulate growth control through the regulation of cyclin D may be related to its cell-survival properties. NF-B is involved in cell cycle regulation by its ability to promote transition from the G1 phase to the S phase in mouse embryonic fibroblasts. NF-B activation during the early phases of the cell cycle is therefore necessary in regulating both growth and differentiation, and, in particular, it has been established that induction of DNA binding by NF-B plays a role in hepatocyte proliferation.

STAT3 has also shown to be important during embryonic development and is expressed in a tissue-restricted manner during embryogenesis. STAT3-deficient mice die during embryogenesis, which indicates that STAT3 plays a crucial role in a variety of biological functions such as cell growth, suppression of apoptosis, and cell motility. STAT3 activity is detected in early post-implantation development in mice, and STAT3 RNA is expressed as early as E7.5. STAT3 is therefore important during early development, and it is possible that PIF plays a role in its activation.

In addition to the importance of NF-B and STAT3 in early development and growth, ICAM-1 expression may also play a role in development. Undifferentiated embryonic stem cells have increased ICAM-1 expression in the presence of leukemia inhibitory factor, a member of the IL-6 family that can also induce STAT3 activation. It has been suggested that the pattern of expression of IgICAMs such as ICAM-1 has a role in defining the phenotype of both differentiated and undifferentiated cells.

These data may suggest that the primary role of PIF is, in fact, during embryonic development, which may account for the differences in effect observed, on adult tissues, between PIF and proinflammatory cytokines in the induction of both cytokines and acute phase proteins. It is possible that PIF may have an important role in the induction of several important developmental transcription factors during embryonic development, which results in the tightly regulated and coordinated responses involved in tissue engineering.

In this study we have shown that PIF can induce NF-B and STAT3 in isolated human hepatocytes with the resultant expression of proinflammatory cytokines, adhesion molecules, and acute phase proteins. PIF may thus play a role in cancer cachexia, in addition to its effects on skeletal muscle, by contributing to a continuous cycle of cytokine and acute-phase protein production. It is also possible, however, that the normal, constitutive role of PIF might be to induce both NF-B and STAT3 to promote cell growth and differentiation during early embryogenesis. This possibility requires further investigation.

View larger version (33K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0534fje ; to cite this article, use FASEB J. (January 19, 2001) 10.1096/fj.00-0534fje

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