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Intracrine hepatopoietin potentiates AP-1 activity through JAB1 independent of MAPK pathway ¹

CHENGRONG LU^*, YONG LI^*, YANLIN ZHAO^*, GUICHUN XING^*, FEI TANG^*, QINGMING WANG^*, YUHUI SUN², HANDONG WEI^*, XIAOMING YANG^*, CHUTSE WU^*, JIANGUO CHEN², KUN-LIANG GUAN^*, CHENGGANG ZHANG^*, HUIPENG CHEN^* and FUCHU HE^*,3

^* Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Chinese National Human Genome Center at Beijing, Beijing 100850, P. R. China; and
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, P. R. China

³Correspondence: Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, P. R. China. E-mail: hefc{at}nic.bmi.ac.cn

SPECIFIC AIMS

Hepatopoietin (HPO) is a specifically hepatotropic factor originally identified from the cytosol of regenerating or hyperplastic hepatic cells. Although previous results showed that extracellular HPO triggers the MAPK signal transduction by binding to its specific receptor on the cell surface, leading to the proliferation of hepatocyte, the intracellular action of HPO is unclear. The aim of the study is to investigate the possible intracellular function or signaling pathway of HPO.

PRINCIPAL FINDINGS

1. HPO interacts with JAB1 in the yeast two-hybrid system
Using the yeast two-hybrid system to screen the HPO-interacting protein(s), we identified several types of clones that interacted specifically with HPO. One contained a cDNA insert with almost the entire coding sequence (amino acids 39–335) of human JAB1 (Jun-activating domain binding protein 1), a coactivator of AP-1. Either JAB1 deletant (amino acids 39–335) or wild-type JAB1 (amino acids 1–335) interacts with HPO similarly. Additional results indicate that amino-terminal region (1–63) of HPO is sufficient for binding to JAB1 whereas the carboxyl-terminal half sequence of HPO (63–125) is not required.

2. HPO interacts specifically with JAB1 in vitro and in vivo
We further verified a specific association between JAB1 and HPO by GST pull-down and coimmunoprecipitation experiments of JAB1-HPO complexes. Recombinant HPO (rHPO) and ectopically expressed HPO (Flag-HPO) in COS7 cells bound to a GST-JAB1 fusion protein, not to GST alone (Fig. 1 a, left and middle). GST-HPO protein, but not GST alone, specifically coprecipitated the endogenous JAB1 protein from cell extracts (Fig. 1a , right). We also observed specific complex formation in vivo between JAB1 and HPO when both proteins were coexpressed in COS7 cells (Fig. 1b ). Endogenous JAB1-HPO complexes were precipitated from nuclear extracts of human fetal liver (Fig. 1c , upper panel) and adult liver tissues (Fig. 1c , lower panel).

View larger version (24K): [in this window] [in a new window]   Figure 1. HPO interacts with JAB1 in vitro and in vivo. a) rHPO and flag-HPO expressed in COS 7 cells were incubated with immobilized GST, GST-JAB1 fusion proteins. Bound proteins were eluted with excess glutathione and immunoblotted with anti-HPO and anti-flag antibodies (left and middle). Extracts of nontransfected cells were incubated with immobilized GST, GST-HPO fusion proteins. Bound proteins were eluted with excess glutathione and immunoblotted with anti-JAB1 antibody (right). b) COS 7 cells were cotransfected with HA-tagged JAB1 and either GFP-tagged HPO or GFP control vector. The cell lysates were incubated with anti-HA antibody in the protein A/G-Sepharose beads. The immuno-complex was analyzed by immunoblotting with anti-GFP antibody. One-tenth of the total cell lysate used in immunoprecipitation was loaded onto lanes 1 and 2 as an indication of the relative expression level for GFP and GFP-HPO. Lanes 4 and 5 are from cells expressing HA-JAB1/GFP and HA-JAB1/GFP-HPO, respectively. Lane 3 is a control for lane 5 with mouse mock antibody (normal IgG). A duplicate blot was probed with anti-HA antibody to monitor the amounts of JAB1 protein precipitated in each reaction (bottom). c) Endogenous HPO-JAB1 complex was immunoprecipitated from nuclear extracts of human fetal liver (upper panel) and human adult liver (lower panel) with anti-JAB1 antibody and HPO was detected by immunoblotting with anti-HPO antibody. Precipitation with mouse mock antibody (normal IgG) served as controls. Two blots were also probed with anti-JAB1 antibody to monitor the amounts of JAB1 precipitate in each reaction (bottoms of upper and lower panels).

3. Similar distribution or colocalization of HPO with JAB1 within cells
The association between HPO and JAB1 was further confirmed in colocalization of the two proteins in vivo. The endogenous HPO was localized in both cytoplasm and nucleus, with a relative stronger staining of the nucleus in HepG2 cells. Similarly, the endogenous JAB1 was localized primarily in the nucleus except in nucleolus. Thus, endogenous HPO and endogenous JAB1 have a similar pattern of distribution or colocalization in HepG2 cells. Transiently expressed GFP-HPO gather in the nuclear periphery of COS 7 cells, inconsistent with the localization of endogenous HPO in HepG2 cells. The localization of transiently expressed RFP-JAB1 in COS 7 cells is also different from the endogenous JAB1 in HepG2 cells. Single RFP-JAB1 localizes in the cytoplasm of COS 7 cells rather in the nucleus. When HPO and JAB1 were cotransfected into COS 7 cells, their distribution was changed. A part of GFP-HPO proteins was diffusely present in the cytoplasm and colocalized with RFP-JAB1 between the two nuclei in a binucleated cell.

4. The physical interaction of HPO with JAB1 enhances AP-1 activity
JAB1 could activate the AP-1-dependent promoter. To study the function of the HPO-JAB1 interaction, we determined whether HPO modulates JAB1-induced AP-1 activity. COS7 cells were transfected with an AP-1-driven luciferase reporter gene in the presence of c-Jun, JAB1, and HPO, and the reporter gene activity was measured. The results indicated that overexpression of JAB1 caused an increase in the relative activation levels of the AP-1 reporter and that expression of HPO further enhanced potentiation of AP-1 reporter gene activity induced by cotransfected JAB1 (Fig. 2 a). HPO appeared to increase JAB1-induced AP-1 activity in a concentration-dependent manner (Fig. 2a ). Ectopical expression of HPO alone could potentiate AP-1 activity, presumably through endogenous JAB1 (Fig. 2b ). To further confirm the role of HPO in potentiation of JAB1 on AP-1 activity, we studied the effect of suppressed expression of JAB1. Overexpression of JAB1 antisense constructs resulted in an obvious reduction of endogenous JAB1 level (Fig. 2c , lower panel) and significantly inhibited activation of the AP-1 reporter gene induced by intracellularly expressed HPO (Fig. 2c , upper panel). The deletants of HPO, i.e., either HPO^1–63 or HPO^63–125, were unable to potentiate AP-1 activity in response to JAB1, although HPO^1–63 was able to bind to JAB1. These results together indicate that JAB1 is involved in the intracellular signaling transduction from HPO to AP-1.

View larger version (35K): [in this window] [in a new window]   Figure 2. Regulation of AP-1 activity by HPO interacting with JAB1. a) COS 7 cells were cotransfected with AP-1-driven luciferase reporter, c-Jun, JAB1, and HPO or its mutants. b) COS 7 cells were cotransfected with AP-1-driven luciferase reporter, c-Jun, and HPO or its mutant in the absence of JAB1 transfection. c) COS7 cells were cotransfected with AP-1-driven luciferase reporter, c-Jun, HPO, and one of the indicated antisense constructs. Lower panel indicates the reduction of endogenous JAB1 by JAB1 antisense or IL-12R ß2 subunit antisense (control antisense). ß-Actin content in cell extracts was immunoblotted as a loading control. Luciferase activity was normalized to a cotransfected ß-galactosidase expression vector. Results are representative of at least three independent experiments performed in duplicate are shown. The error bar indicates SD.

5. JAB1 increases phospho-c-Jun levels and intracrine HPO enhances this effect of JAB1
It was demonstrated that JAB1 could increase phosphorylation of c-Jun and HPO further enhance this function of JAB1. These results conform to the data of an AP-1 reporter gene activation assay (Fig. 2a ). However, neither JAB1 nor HPO had any effect on the expression of transfected c-Jun, endogenous JNK, or its phosphorylation. These data showed that c-Jun in HPO intracellular signaling may be phosphorylated via a JNK-independent pathway.

Our previous data indicate that extracellular HPO triggers MAPK/ERK pathway. In this study, however, we excluded the possible contribution of MAPK signaling pathway to the activation of AP-1 activity induced by HPO under mediation of JAB1. Taken together, these data indicate that HPO interacts with JAB1 to activate AP-1 transcription activity by potentiating phosphorylation of c-Jun in a MAPK-independent fashion.

CONCLUSIONS

Cytokines and growth factors with mitogenic effects induce potentiation of AP-1 trans-activation function by triggering several signal transduction pathways. Our data showed that intracrine HPO interacts with JAB1 in vivo to regulate AP-1 transcriptional activity, indicating a new mechanism for AP-1 activation by cytokines and growth factors. To our knowledge, HPO is the first intracrine growth factor identified to trigger AP-1 pathway through intracellular interaction with a transcriptional coactivator. Intracrine HPO with JAB1 represents a new intracellular short-cut signaling pathway that could provide a novel mechanism in triggering immediate-early response transcription factors during initiation of liver regeneration. The ERV/HPO family has thiol oxidoreductase to participate in a cytoplasmic pathway of disulfide bond formation. Increasing evidence has indicated that cellular redox status modulates various aspects of cellular events such as proliferation and apoptosis wherein redox regulation of transcription factors (including AP-1, NF-B, Myb, and Ets) is an important issue. Therefore, regulation of the JAB1 function or AP-1 transcriptional activity by HPO may be associated with the redox enzyme activity.

JAB1 is a subunit of COP9 complex containing at least eight subunits. The plant COP9 is involved in light-mediated signal transduction. COP9 is reported to have kinase activity that phosphorylates IB and c-Jun. Our data implicate that HPO might increase the phosphorylation of c-Jun under mediation of JAB1-containing COP9 signalosome, leading to activation of AP-1 activity. JAB1 is regulated by different proteins in order to redistribute within cells and undergo different functions. For example, after interaction with integrin LFA-1, which transduces signals, JAB1 moves from cytoplasm to nucleus to induce AP-1 activation. JAB1 can also interact with p27kip1 in the nucleus and direct its movement to the cytoplasm for protein breakdown. Apparently JAB1 is a protein with multifunctional characteristics. We found colocalization of HPO with JAB1 in the nucleus. It can be speculated that the COP9 complex could represent an intracellular signal crossroad, where signals from the extracellular or intracellular environment are coordinated with transcriptional activation and regulation of related cell functions.

Because the specific receptor of HPO exists only on the surface of liver cells, the action of HPO to trigger MAP kinase pathway might be liver specific. However, the intracellular function of HPO to stimulate AP-1 activity through JAB1 indicates a new pathway whereby HPO signaling regulates gene expression, which may contribute to the physiological regulation of HPO-originated tissues such as testis, kidney, brain, and liver. Whether the dual signaling pathways of HPO (Fig. 3 ) cooperate with each other or act separately remains to be elucidated. It is possible that extracellular HPO secreted as an autocrine growth factor maintains the autonomous growth of liver cells and intracellular HPO, as an intracrine factor triggers AP-1 pathway by regulation of JAB1 for immediate-early response when its intracellular level is increased after partial liver hepatectomy or liver injury. Rapid up-regulation of HPO expression and AP-1 activity during liver regeneration might support this hypothesis, which would be a valid subject for further investigation.

View larger version (10K): [in this window] [in a new window]   Figure 3. A model of the dual signaling pathways of HPO. Extracellular HPO autocrined from hepatocyte or hepatoma cells activates the MAPK signaling pathway via binding to the receptor. Intracellular HPO activates AP-1 activity under the mediation of JAB1.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0506fje; to cite this article, use FASEB J. (November 14, 2001) 10.1096/fj.01-0506fje

² Present address: Peking University School of Life Sciences, Beijing 100871, P. R. China.

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