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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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Time and dose-dependent induction of RPTP-κ by TGF-β in primary human keratinocytes. Keratinocytes were treated with vehicle (0) or TGF-β1 (2.5 ng/ml) for indicated times (a and b), or for 24 h with the indicated concentrations of TGF-β (c and d). (a and c) Total RNA was isolated and RPTP-κ and 36B4 (internal control for normalization) mRNA levels were determined by real-time RT-PCR analyses. n = 3, *p < 0.05. (b and d) Equal amounts of whole cell lysates were analyzed for RPTP-κ and β-actin (internal control for normalization) protein levels by Western blots, which were quantified by chemifluorescence, using a STORM PhosphorImager. Insets, representative Western blots. n = 3, *p < 0.05.
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Figure 1: Time and dose-dependent induction of RPTP-κ by TGF-β in primary human keratinocytes. Keratinocytes were treated with vehicle (0) or TGF-β1 (2.5 ng/ml) for indicated times (a and b), or for 24 h with the indicated concentrations of TGF-β (c and d). (a and c) Total RNA was isolated and RPTP-κ and 36B4 (internal control for normalization) mRNA levels were determined by real-time RT-PCR analyses. n = 3, *p < 0.05. (b and d) Equal amounts of whole cell lysates were analyzed for RPTP-κ and β-actin (internal control for normalization) protein levels by Western blots, which were quantified by chemifluorescence, using a STORM PhosphorImager. Insets, representative Western blots. n = 3, *p < 0.05.

Mentions: We initially examined kinetics and dose dependence of induction of RPTP-κ by TGF-β and its functional consequences on EGFR activation. TGF-β induced RPTP-κ mRNA (Figure 1a) and protein (Figure 1b) levels, in a time-dependent manner. Induction of RPTP-κ by TGF-β was observed at 3 h, was maximal within 24 h, and remained maximally elevated for at least 48 h. Maximal induction of RPTP-κ mRNA (Figure 1c) and protein (Figure 1d) was observed at 1.3 ng/ml TGF-β1. Higher concentrations of TGF-β1 reduced RPTP-κ protein levels, but not mRNA levels, relative to maximal stimulation achieved by 1.3 μg/ml TGF-β1, suggesting posttranslational regulation of RPTP-κ protein expression.


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)

Time and dose-dependent induction of RPTP-κ by TGF-β in primary human keratinocytes. Keratinocytes were treated with vehicle (0) or TGF-β1 (2.5 ng/ml) for indicated times (a and b), or for 24 h with the indicated concentrations of TGF-β (c and d). (a and c) Total RNA was isolated and RPTP-κ and 36B4 (internal control for normalization) mRNA levels were determined by real-time RT-PCR analyses. n = 3, *p < 0.05. (b and d) Equal amounts of whole cell lysates were analyzed for RPTP-κ and β-actin (internal control for normalization) protein levels by Western blots, which were quantified by chemifluorescence, using a STORM PhosphorImager. Insets, representative Western blots. n = 3, *p < 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2801716&req=5

Figure 1: Time and dose-dependent induction of RPTP-κ by TGF-β in primary human keratinocytes. Keratinocytes were treated with vehicle (0) or TGF-β1 (2.5 ng/ml) for indicated times (a and b), or for 24 h with the indicated concentrations of TGF-β (c and d). (a and c) Total RNA was isolated and RPTP-κ and 36B4 (internal control for normalization) mRNA levels were determined by real-time RT-PCR analyses. n = 3, *p < 0.05. (b and d) Equal amounts of whole cell lysates were analyzed for RPTP-κ and β-actin (internal control for normalization) protein levels by Western blots, which were quantified by chemifluorescence, using a STORM PhosphorImager. Insets, representative Western blots. n = 3, *p < 0.05.
Mentions: We initially examined kinetics and dose dependence of induction of RPTP-κ by TGF-β and its functional consequences on EGFR activation. TGF-β induced RPTP-κ mRNA (Figure 1a) and protein (Figure 1b) levels, in a time-dependent manner. Induction of RPTP-κ by TGF-β was observed at 3 h, was maximal within 24 h, and remained maximally elevated for at least 48 h. Maximal induction of RPTP-κ mRNA (Figure 1c) and protein (Figure 1d) was observed at 1.3 ng/ml TGF-β1. Higher concentrations of TGF-β1 reduced RPTP-κ protein levels, but not mRNA levels, relative to maximal stimulation achieved by 1.3 μg/ml TGF-β1, suggesting posttranslational regulation of RPTP-κ protein expression.

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