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Receptor type protein tyrosine phosphatase-kappa mediates cross-talk between transforming growth factor-beta and epidermal growth factor receptor signaling pathways in human keratinocytes.

Xu Y, Baker D, Quan T, Baldassare JJ, Voorhees JJ, Fisher GJ - Mol. Biol. Cell (2009)

Bottom Line: Induction of RPTP-kappa by TGF-beta significantly decreases basal and EGF-stimulated EGFR tyrosine phosphorylation. shRNA-mediated reduction of TGF-beta-induced RPTP-kappa significantly attenuates the ability of TGF-beta to inhibit proliferation.Smad3/4 binding is localized to an 186-base pair region, which contains a consensus Smad3-binding element.These data describe a novel mechanism of cross-talk between EGFR and TGF-beta pathways, in which RPTP-kappa functions to integrate growth-promoting and growth-inhibiting signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Michigan, Ann Arbor, MI 48109-5609, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) signaling pathways promote human keratinocyte survival and proliferation. In contrast, transforming growth factor-beta (TGF-beta) signaling pathways are strongly anti-proliferative. Receptor type protein tyrosine phosphatase-kappa (RPTP-kappa) specifically dephosphorylates EGFR, thereby blocking EGFR-dependent signaling, and inhibiting proliferation. We report here that RPTP-kappa mediates functional integration of EGFR and TGF-beta signaling pathways in human keratinocytes. TGF-beta up-regulates RPTP-kappa mRNA and protein, in a dose and time dependent manner. Induction of RPTP-kappa by TGF-beta significantly decreases basal and EGF-stimulated EGFR tyrosine phosphorylation. shRNA-mediated reduction of TGF-beta-induced RPTP-kappa significantly attenuates the ability of TGF-beta to inhibit proliferation. RPTP-kappa induction is dependent on activation of transcription factors Smad3 and Smad4. Inhibition of TGF-beta receptor kinase completely prevents induction of RPTP-kappa. Chromatin immunoprecipitation assays reveal that TGF-beta stimulates Smad3 and Smad4 binding to RPTP-kappa gene promoter. Smad3/4 binding is localized to an 186-base pair region, which contains a consensus Smad3-binding element. These data describe a novel mechanism of cross-talk between EGFR and TGF-beta pathways, in which RPTP-kappa functions to integrate growth-promoting and growth-inhibiting signaling pathways.

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TGF-β promotes binding of Smad3 and Smad4 to the RPTP-κ promoter in primary human keratinocytes. Keratinocytes were treated with vehicle (open bar) or TGF-β1 (closed bar, 2.5 ng/ml) for 3 h. Cross-linked and sheared chromosomal DNA was immunoprecipitated with (a) Smad4 or (b) Smad3 antibody. Immunoprecipitation with IgG was used as control. Immunoprecipitated DNA was amplified by real-time PCR, using four pairs of primers that covered proximal 1-kb region of human RPTP-κ promoter. Data are means; error bars, SEM; n = 3; *p < 0.05.
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Figure 7: TGF-β promotes binding of Smad3 and Smad4 to the RPTP-κ promoter in primary human keratinocytes. Keratinocytes were treated with vehicle (open bar) or TGF-β1 (closed bar, 2.5 ng/ml) for 3 h. Cross-linked and sheared chromosomal DNA was immunoprecipitated with (a) Smad4 or (b) Smad3 antibody. Immunoprecipitation with IgG was used as control. Immunoprecipitated DNA was amplified by real-time PCR, using four pairs of primers that covered proximal 1-kb region of human RPTP-κ promoter. Data are means; error bars, SEM; n = 3; *p < 0.05.

Mentions: The data presented above suggest that Smad3/4 may directly regulate RPTP-κ gene transcription. To further substantiate this conclusion, we performed ChIP assay, to determine Smad3/4 binding to the RPTP-κ gene promoter. Human primary keratinocytes were treated with vehicle or TGF-β for 3 h, and cross-linked, sheered chromatin was immunoprecipitated with either Smad3 or Smad4 antibodies. Immunoprecipitated chromatin DNA was amplified using four pairs of primer sets covering the proximal 1-kb region of human RPTP-κ promoter. As shown in Figures 7, a and b, TGF-β specifically induced binding of both Smad3 and Smad4 to a region of the RPTP-κ promoter located between nucleotides 522 and 706. This region of the RPTP-κ promoter contains a GTCT sequence, which closely conforms to the consensus Smad3-binding sequence (Yagi et al., 2002). These data indicate that regulation of RPTP-κ gene expression by TGF-β-activated Smad3/4 (Figure 7) is mediated by direct binding of Smad3/4 to the RPTP-κ promoter.


Receptor type protein tyrosine phosphatase-kappa mediates cross-talk between transforming growth factor-beta and epidermal growth factor receptor signaling pathways in human keratinocytes.

Xu Y, Baker D, Quan T, Baldassare JJ, Voorhees JJ, Fisher GJ - Mol. Biol. Cell (2009)

TGF-β promotes binding of Smad3 and Smad4 to the RPTP-κ promoter in primary human keratinocytes. Keratinocytes were treated with vehicle (open bar) or TGF-β1 (closed bar, 2.5 ng/ml) for 3 h. Cross-linked and sheared chromosomal DNA was immunoprecipitated with (a) Smad4 or (b) Smad3 antibody. Immunoprecipitation with IgG was used as control. Immunoprecipitated DNA was amplified by real-time PCR, using four pairs of primers that covered proximal 1-kb region of human RPTP-κ promoter. Data are means; error bars, SEM; n = 3; *p < 0.05.
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Related In: Results  -  Collection

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Figure 7: TGF-β promotes binding of Smad3 and Smad4 to the RPTP-κ promoter in primary human keratinocytes. Keratinocytes were treated with vehicle (open bar) or TGF-β1 (closed bar, 2.5 ng/ml) for 3 h. Cross-linked and sheared chromosomal DNA was immunoprecipitated with (a) Smad4 or (b) Smad3 antibody. Immunoprecipitation with IgG was used as control. Immunoprecipitated DNA was amplified by real-time PCR, using four pairs of primers that covered proximal 1-kb region of human RPTP-κ promoter. Data are means; error bars, SEM; n = 3; *p < 0.05.
Mentions: The data presented above suggest that Smad3/4 may directly regulate RPTP-κ gene transcription. To further substantiate this conclusion, we performed ChIP assay, to determine Smad3/4 binding to the RPTP-κ gene promoter. Human primary keratinocytes were treated with vehicle or TGF-β for 3 h, and cross-linked, sheered chromatin was immunoprecipitated with either Smad3 or Smad4 antibodies. Immunoprecipitated chromatin DNA was amplified using four pairs of primer sets covering the proximal 1-kb region of human RPTP-κ promoter. As shown in Figures 7, a and b, TGF-β specifically induced binding of both Smad3 and Smad4 to a region of the RPTP-κ promoter located between nucleotides 522 and 706. This region of the RPTP-κ promoter contains a GTCT sequence, which closely conforms to the consensus Smad3-binding sequence (Yagi et al., 2002). These data indicate that regulation of RPTP-κ gene expression by TGF-β-activated Smad3/4 (Figure 7) is mediated by direct binding of Smad3/4 to the RPTP-κ promoter.

Bottom Line: Induction of RPTP-kappa by TGF-beta significantly decreases basal and EGF-stimulated EGFR tyrosine phosphorylation. shRNA-mediated reduction of TGF-beta-induced RPTP-kappa significantly attenuates the ability of TGF-beta to inhibit proliferation.Smad3/4 binding is localized to an 186-base pair region, which contains a consensus Smad3-binding element.These data describe a novel mechanism of cross-talk between EGFR and TGF-beta pathways, in which RPTP-kappa functions to integrate growth-promoting and growth-inhibiting signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Michigan, Ann Arbor, MI 48109-5609, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) signaling pathways promote human keratinocyte survival and proliferation. In contrast, transforming growth factor-beta (TGF-beta) signaling pathways are strongly anti-proliferative. Receptor type protein tyrosine phosphatase-kappa (RPTP-kappa) specifically dephosphorylates EGFR, thereby blocking EGFR-dependent signaling, and inhibiting proliferation. We report here that RPTP-kappa mediates functional integration of EGFR and TGF-beta signaling pathways in human keratinocytes. TGF-beta up-regulates RPTP-kappa mRNA and protein, in a dose and time dependent manner. Induction of RPTP-kappa by TGF-beta significantly decreases basal and EGF-stimulated EGFR tyrosine phosphorylation. shRNA-mediated reduction of TGF-beta-induced RPTP-kappa significantly attenuates the ability of TGF-beta to inhibit proliferation. RPTP-kappa induction is dependent on activation of transcription factors Smad3 and Smad4. Inhibition of TGF-beta receptor kinase completely prevents induction of RPTP-kappa. Chromatin immunoprecipitation assays reveal that TGF-beta stimulates Smad3 and Smad4 binding to RPTP-kappa gene promoter. Smad3/4 binding is localized to an 186-base pair region, which contains a consensus Smad3-binding element. These data describe a novel mechanism of cross-talk between EGFR and TGF-beta pathways, in which RPTP-kappa functions to integrate growth-promoting and growth-inhibiting signaling pathways.

Show MeSH
Related in: MedlinePlus