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Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation.

Yoshida T, Hanada T, Tokuhisa T, Kosai K, Sata M, Kohara M, Yoshimura A - J. Exp. Med. (2002)

Bottom Line: Activation of STAT3 by the HCV core in NIH-3T3 cells resulted in rapid proliferation and up-regulation of Bcl-XL and cyclin-D1.Additional expression of STAT3 in HCV core-expressing cells resulted in anchorage-independent growth and tumorigenesis.We propose that the HCV core protein cooperates with STAT3, which leads to cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

ABSTRACT
The signal transducer and activator of transcription (STAT) family proteins are transcription factors critical in mediating cytokine signaling. Among them, STAT3 is often constitutively phosphorylated and activated in human cancers and in transformed cell lines and is implicated in tumorigenesis. However, cause of the persistent activation of STAT3 in human tumor cells is largely unknown. The hepatitis C virus (HCV) is a major etiological agent of non-A and non-B hepatitis, and chronic infection by HCV is associated with development of liver cirrhosis and hepatocellular carcinoma. HCV core protein is proposed to be responsible for the virus-induced transformation. We now report that HCV core protein directly interacts with and activates STAT3 through phosphorylation of the critical tyrosine residue. Activation of STAT3 by the HCV core in NIH-3T3 cells resulted in rapid proliferation and up-regulation of Bcl-XL and cyclin-D1. Additional expression of STAT3 in HCV core-expressing cells resulted in anchorage-independent growth and tumorigenesis. We propose that the HCV core protein cooperates with STAT3, which leads to cellular transformation.

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Interaction between the core protein and STAT3. (A) In vitro binding experiment. HepG2 cell extracts stimulated with (+) or without (−) LIF were incubated with either GST or GST-core bound to glutathione-Sepharose. Aliquots of total cell extracts (TCL) and proteins bound to the beads were immunoblotted with anti-STAT3, anti-NF-κB p65, and anti-ERK2 antibodies. Purified GST and GST-core fusion protein (0.1 μg protein) was also blotted with anti-GST. (B) Schematic diagrams of structural domains and deletion mutants of STAT3. (C) Flag-tagged STAT3 deletion mutants and the myc-tagged core were coexpressed in 293 cells and then immunoprecipitated with either anti-Myc or anti-Flag antibodies. The total cell extracts (TCL) or immunoprecipitates (IP) were blotted with anti-Myc or anti-Flag antibodies. To confirm the lack of binding between the core protein and ΔL-TA construct, data of a separate experiment are shown in the right (exp. 2). (D) Schematic diagrams of the deletion construct of the core protein. (E) HepG2 cells were transfected with an APRE-luciferase reporter gene and an increased amount of indicated core deletion mutants. After 24 h transfection, luciferase activity was measured. (F) The 293 cells were transfected with flag-tagged STAT3 and myc-tagged core deletion mutants. Immunoprecipitates with anti-Myc antibody or TCL were blotted with anti-Flag and anti-Myc antibodies.
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fig3: Interaction between the core protein and STAT3. (A) In vitro binding experiment. HepG2 cell extracts stimulated with (+) or without (−) LIF were incubated with either GST or GST-core bound to glutathione-Sepharose. Aliquots of total cell extracts (TCL) and proteins bound to the beads were immunoblotted with anti-STAT3, anti-NF-κB p65, and anti-ERK2 antibodies. Purified GST and GST-core fusion protein (0.1 μg protein) was also blotted with anti-GST. (B) Schematic diagrams of structural domains and deletion mutants of STAT3. (C) Flag-tagged STAT3 deletion mutants and the myc-tagged core were coexpressed in 293 cells and then immunoprecipitated with either anti-Myc or anti-Flag antibodies. The total cell extracts (TCL) or immunoprecipitates (IP) were blotted with anti-Myc or anti-Flag antibodies. To confirm the lack of binding between the core protein and ΔL-TA construct, data of a separate experiment are shown in the right (exp. 2). (D) Schematic diagrams of the deletion construct of the core protein. (E) HepG2 cells were transfected with an APRE-luciferase reporter gene and an increased amount of indicated core deletion mutants. After 24 h transfection, luciferase activity was measured. (F) The 293 cells were transfected with flag-tagged STAT3 and myc-tagged core deletion mutants. Immunoprecipitates with anti-Myc antibody or TCL were blotted with anti-Flag and anti-Myc antibodies.

Mentions: Activation of STAT3 by the core protein suggests a direct interaction between these two proteins. As shown in Fig. 3 , STAT3 bound to the core both in vitro and in vivo. Both phosphorylated and nonphosphorylated forms of STAT3 bound to the GST-fused core protein in vitro (Fig. 3 A). We examined whether the core protein directly interacts with ERK2 (MAP kinase) or the NF-κB p65 subunit, as the core has been shown to activate MAP kinase and NF-κB under certain circumstances (14, 16). We observed no direct interaction of the core with these proteins in vitro (Fig. 3 A).


Activation of STAT3 by the hepatitis C virus core protein leads to cellular transformation.

Yoshida T, Hanada T, Tokuhisa T, Kosai K, Sata M, Kohara M, Yoshimura A - J. Exp. Med. (2002)

Interaction between the core protein and STAT3. (A) In vitro binding experiment. HepG2 cell extracts stimulated with (+) or without (−) LIF were incubated with either GST or GST-core bound to glutathione-Sepharose. Aliquots of total cell extracts (TCL) and proteins bound to the beads were immunoblotted with anti-STAT3, anti-NF-κB p65, and anti-ERK2 antibodies. Purified GST and GST-core fusion protein (0.1 μg protein) was also blotted with anti-GST. (B) Schematic diagrams of structural domains and deletion mutants of STAT3. (C) Flag-tagged STAT3 deletion mutants and the myc-tagged core were coexpressed in 293 cells and then immunoprecipitated with either anti-Myc or anti-Flag antibodies. The total cell extracts (TCL) or immunoprecipitates (IP) were blotted with anti-Myc or anti-Flag antibodies. To confirm the lack of binding between the core protein and ΔL-TA construct, data of a separate experiment are shown in the right (exp. 2). (D) Schematic diagrams of the deletion construct of the core protein. (E) HepG2 cells were transfected with an APRE-luciferase reporter gene and an increased amount of indicated core deletion mutants. After 24 h transfection, luciferase activity was measured. (F) The 293 cells were transfected with flag-tagged STAT3 and myc-tagged core deletion mutants. Immunoprecipitates with anti-Myc antibody or TCL were blotted with anti-Flag and anti-Myc antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194001&req=5

fig3: Interaction between the core protein and STAT3. (A) In vitro binding experiment. HepG2 cell extracts stimulated with (+) or without (−) LIF were incubated with either GST or GST-core bound to glutathione-Sepharose. Aliquots of total cell extracts (TCL) and proteins bound to the beads were immunoblotted with anti-STAT3, anti-NF-κB p65, and anti-ERK2 antibodies. Purified GST and GST-core fusion protein (0.1 μg protein) was also blotted with anti-GST. (B) Schematic diagrams of structural domains and deletion mutants of STAT3. (C) Flag-tagged STAT3 deletion mutants and the myc-tagged core were coexpressed in 293 cells and then immunoprecipitated with either anti-Myc or anti-Flag antibodies. The total cell extracts (TCL) or immunoprecipitates (IP) were blotted with anti-Myc or anti-Flag antibodies. To confirm the lack of binding between the core protein and ΔL-TA construct, data of a separate experiment are shown in the right (exp. 2). (D) Schematic diagrams of the deletion construct of the core protein. (E) HepG2 cells were transfected with an APRE-luciferase reporter gene and an increased amount of indicated core deletion mutants. After 24 h transfection, luciferase activity was measured. (F) The 293 cells were transfected with flag-tagged STAT3 and myc-tagged core deletion mutants. Immunoprecipitates with anti-Myc antibody or TCL were blotted with anti-Flag and anti-Myc antibodies.
Mentions: Activation of STAT3 by the core protein suggests a direct interaction between these two proteins. As shown in Fig. 3 , STAT3 bound to the core both in vitro and in vivo. Both phosphorylated and nonphosphorylated forms of STAT3 bound to the GST-fused core protein in vitro (Fig. 3 A). We examined whether the core protein directly interacts with ERK2 (MAP kinase) or the NF-κB p65 subunit, as the core has been shown to activate MAP kinase and NF-κB under certain circumstances (14, 16). We observed no direct interaction of the core with these proteins in vitro (Fig. 3 A).

Bottom Line: Activation of STAT3 by the HCV core in NIH-3T3 cells resulted in rapid proliferation and up-regulation of Bcl-XL and cyclin-D1.Additional expression of STAT3 in HCV core-expressing cells resulted in anchorage-independent growth and tumorigenesis.We propose that the HCV core protein cooperates with STAT3, which leads to cellular transformation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

ABSTRACT
The signal transducer and activator of transcription (STAT) family proteins are transcription factors critical in mediating cytokine signaling. Among them, STAT3 is often constitutively phosphorylated and activated in human cancers and in transformed cell lines and is implicated in tumorigenesis. However, cause of the persistent activation of STAT3 in human tumor cells is largely unknown. The hepatitis C virus (HCV) is a major etiological agent of non-A and non-B hepatitis, and chronic infection by HCV is associated with development of liver cirrhosis and hepatocellular carcinoma. HCV core protein is proposed to be responsible for the virus-induced transformation. We now report that HCV core protein directly interacts with and activates STAT3 through phosphorylation of the critical tyrosine residue. Activation of STAT3 by the HCV core in NIH-3T3 cells resulted in rapid proliferation and up-regulation of Bcl-XL and cyclin-D1. Additional expression of STAT3 in HCV core-expressing cells resulted in anchorage-independent growth and tumorigenesis. We propose that the HCV core protein cooperates with STAT3, which leads to cellular transformation.

Show MeSH
Related in: MedlinePlus