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Modulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phosphatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor.

You M, Flick LM, Yu D, Feng GS - J. Exp. Med. (2001)

Bottom Line: The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis.In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed.These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Shp-2, a src homology (SH)2-containing phosphotyrosine phosphatase, appears to be involved in cytoplasmic signaling downstream of a variety of cell surface receptors, although the mechanism is unclear. Here, we have determined a role of Shp-2 in the cytokine circuit for inflammatory and immune responses. Production of interleukin (IL)-6 in response to IL-1 alpha or tumor necrosis factor (TNF)-alpha was nearly abolished in homozygous mutant (Shp-2(-/)-) fibroblast cells. The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis. In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed. Reintroduction of a wild-type Shp-2 protein into Shp-2(-/)- cells rescued NF-kappa B activation and IL-6 production in response to IL-1/TNF stimulation. Furthermore, Shp-2 tyrosine phosphatase was detected in complexes with IKK as well as with IL-1 receptor. Thus, this SH2-containing enzyme is an important cytoplasmic factor required for efficient NF-kappa B activation. These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.

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Rescue of the mutant phenotype by reintroduction of wild-type Shp-2. Shp-2−/− cells were transfected with a pcDNA3.1 vector or a wild-type Shp-2 cDNA expression construct, and clones expressing wild-type Shp-2 were isolated (reference 21). Shp-2−/− cell lines transfected with the vector only (V) or expressing wild-type Shp-2 (R4) were treated with 10 ng/ml IL-1α or 20 ng/ml TNF-α for the indicated time periods. (A and B) The amounts of secreted IL-6 in supernatants were determined by ELISA as described in Fig. 1. Data shown are the means of three independent experiments ± SD. (C) Nuclear extracts were prepared for detection of NF-κB activity as described in Fig. 4. The arrows denote the specific NF-κB–DNA complexes. (D) IkBα phosphorylation was evaluated by anti–P-IκBα antibody blotting.
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Figure 8: Rescue of the mutant phenotype by reintroduction of wild-type Shp-2. Shp-2−/− cells were transfected with a pcDNA3.1 vector or a wild-type Shp-2 cDNA expression construct, and clones expressing wild-type Shp-2 were isolated (reference 21). Shp-2−/− cell lines transfected with the vector only (V) or expressing wild-type Shp-2 (R4) were treated with 10 ng/ml IL-1α or 20 ng/ml TNF-α for the indicated time periods. (A and B) The amounts of secreted IL-6 in supernatants were determined by ELISA as described in Fig. 1. Data shown are the means of three independent experiments ± SD. (C) Nuclear extracts were prepared for detection of NF-κB activity as described in Fig. 4. The arrows denote the specific NF-κB–DNA complexes. (D) IkBα phosphorylation was evaluated by anti–P-IκBα antibody blotting.

Mentions: To confirm that the defect in IL-1/TNF-induced NF-κB activation and IL-6 production were caused by absence of a functional Shp-2, we reevaluated cellular responses to the cytokine stimulation in Shp-2−/− cells in which a wild-type Shp-2 was reintroduced. As illustrated in Fig. 8, reintroduction of wild-type Shp-2 protein rescued the activation of NF-κB by IL-1α or TNF-α. Furthermore, expression of wild-type Shp-2 in mutant cells resulted in a significant increase of IL-6 secretion (Fig. 8).


Modulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phosphatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor.

You M, Flick LM, Yu D, Feng GS - J. Exp. Med. (2001)

Rescue of the mutant phenotype by reintroduction of wild-type Shp-2. Shp-2−/− cells were transfected with a pcDNA3.1 vector or a wild-type Shp-2 cDNA expression construct, and clones expressing wild-type Shp-2 were isolated (reference 21). Shp-2−/− cell lines transfected with the vector only (V) or expressing wild-type Shp-2 (R4) were treated with 10 ng/ml IL-1α or 20 ng/ml TNF-α for the indicated time periods. (A and B) The amounts of secreted IL-6 in supernatants were determined by ELISA as described in Fig. 1. Data shown are the means of three independent experiments ± SD. (C) Nuclear extracts were prepared for detection of NF-κB activity as described in Fig. 4. The arrows denote the specific NF-κB–DNA complexes. (D) IkBα phosphorylation was evaluated by anti–P-IκBα antibody blotting.
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Related In: Results  -  Collection

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Figure 8: Rescue of the mutant phenotype by reintroduction of wild-type Shp-2. Shp-2−/− cells were transfected with a pcDNA3.1 vector or a wild-type Shp-2 cDNA expression construct, and clones expressing wild-type Shp-2 were isolated (reference 21). Shp-2−/− cell lines transfected with the vector only (V) or expressing wild-type Shp-2 (R4) were treated with 10 ng/ml IL-1α or 20 ng/ml TNF-α for the indicated time periods. (A and B) The amounts of secreted IL-6 in supernatants were determined by ELISA as described in Fig. 1. Data shown are the means of three independent experiments ± SD. (C) Nuclear extracts were prepared for detection of NF-κB activity as described in Fig. 4. The arrows denote the specific NF-κB–DNA complexes. (D) IkBα phosphorylation was evaluated by anti–P-IκBα antibody blotting.
Mentions: To confirm that the defect in IL-1/TNF-induced NF-κB activation and IL-6 production were caused by absence of a functional Shp-2, we reevaluated cellular responses to the cytokine stimulation in Shp-2−/− cells in which a wild-type Shp-2 was reintroduced. As illustrated in Fig. 8, reintroduction of wild-type Shp-2 protein rescued the activation of NF-κB by IL-1α or TNF-α. Furthermore, expression of wild-type Shp-2 in mutant cells resulted in a significant increase of IL-6 secretion (Fig. 8).

Bottom Line: The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis.In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed.These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Shp-2, a src homology (SH)2-containing phosphotyrosine phosphatase, appears to be involved in cytoplasmic signaling downstream of a variety of cell surface receptors, although the mechanism is unclear. Here, we have determined a role of Shp-2 in the cytokine circuit for inflammatory and immune responses. Production of interleukin (IL)-6 in response to IL-1 alpha or tumor necrosis factor (TNF)-alpha was nearly abolished in homozygous mutant (Shp-2(-/)-) fibroblast cells. The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis. In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed. Reintroduction of a wild-type Shp-2 protein into Shp-2(-/)- cells rescued NF-kappa B activation and IL-6 production in response to IL-1/TNF stimulation. Furthermore, Shp-2 tyrosine phosphatase was detected in complexes with IKK as well as with IL-1 receptor. Thus, this SH2-containing enzyme is an important cytoplasmic factor required for efficient NF-kappa B activation. These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.

Show MeSH
Related in: MedlinePlus