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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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Knockdown of RPTP-κ reduces TGF-β suppression of proliferation in primary human keratinocytes. Keratinocytes were infected with nontargeting control (Ctrl shRNA) or RPTP-κ shRNA lentivirus, coding puromycin resistance. Cells were cultured for 2 d in the presence of puromycin (2 μg/ml), before addition of vehicle (Ctrl) or TGF-β1 (2.5 ng/ml) for 2 d. Cell numbers were obtained by counting the cells using a hemocytometer. Data are means; error bars, SEM; n = 4; *p <0.05 versus Ctrl.
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Figure 4: Knockdown of RPTP-κ reduces TGF-β suppression of proliferation in primary human keratinocytes. Keratinocytes were infected with nontargeting control (Ctrl shRNA) or RPTP-κ shRNA lentivirus, coding puromycin resistance. Cells were cultured for 2 d in the presence of puromycin (2 μg/ml), before addition of vehicle (Ctrl) or TGF-β1 (2.5 ng/ml) for 2 d. Cell numbers were obtained by counting the cells using a hemocytometer. Data are means; error bars, SEM; n = 4; *p <0.05 versus Ctrl.

Mentions: As shown in Figure 3, inhibition of EGFR function by specific tyrosine kinase inhibitors AG1478 (Fry et al., 1994) or PD169540 (Fry et al., 1998) or addition of TGF-β substantially decreases proliferation of primary adult human keratinocytes. Therefore, we next investigated the role of RPTP-κ in TGF-β–mediated inhibition of human primary keratinocyte growth. Cells were infected with lentivirus carrying either a nontargeting control shRNA or a RPTP-κ–targeting shRNA construct (Figure 4), which also carried a puromycin resistance gene. Cultures were treated with puromycin for 2 d to ensure that the majority of cells were expressing the lentivirus constructs. Puromycin-resistant keratinocyte cultures were treated with vehicle or TGF-β1. TGF-β inhibited control cells proliferation 75%, whereas proliferation of RPTP-κ knockdown cells was reduced only 25% (Figure 4). These data indicate that induction of RPTP-κ mediates, in part, the ability of TGF-β to inhibit human keratinocyte proliferation.


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)

Knockdown of RPTP-κ reduces TGF-β suppression of proliferation in primary human keratinocytes. Keratinocytes were infected with nontargeting control (Ctrl shRNA) or RPTP-κ shRNA lentivirus, coding puromycin resistance. Cells were cultured for 2 d in the presence of puromycin (2 μg/ml), before addition of vehicle (Ctrl) or TGF-β1 (2.5 ng/ml) for 2 d. Cell numbers were obtained by counting the cells using a hemocytometer. Data are means; error bars, SEM; n = 4; *p <0.05 versus Ctrl.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801716&req=5

Figure 4: Knockdown of RPTP-κ reduces TGF-β suppression of proliferation in primary human keratinocytes. Keratinocytes were infected with nontargeting control (Ctrl shRNA) or RPTP-κ shRNA lentivirus, coding puromycin resistance. Cells were cultured for 2 d in the presence of puromycin (2 μg/ml), before addition of vehicle (Ctrl) or TGF-β1 (2.5 ng/ml) for 2 d. Cell numbers were obtained by counting the cells using a hemocytometer. Data are means; error bars, SEM; n = 4; *p <0.05 versus Ctrl.
Mentions: As shown in Figure 3, inhibition of EGFR function by specific tyrosine kinase inhibitors AG1478 (Fry et al., 1994) or PD169540 (Fry et al., 1998) or addition of TGF-β substantially decreases proliferation of primary adult human keratinocytes. Therefore, we next investigated the role of RPTP-κ in TGF-β–mediated inhibition of human primary keratinocyte growth. Cells were infected with lentivirus carrying either a nontargeting control shRNA or a RPTP-κ–targeting shRNA construct (Figure 4), which also carried a puromycin resistance gene. Cultures were treated with puromycin for 2 d to ensure that the majority of cells were expressing the lentivirus constructs. Puromycin-resistant keratinocyte cultures were treated with vehicle or TGF-β1. TGF-β inhibited control cells proliferation 75%, whereas proliferation of RPTP-κ knockdown cells was reduced only 25% (Figure 4). These data indicate that induction of RPTP-κ mediates, in part, the ability of TGF-β to inhibit human keratinocyte proliferation.

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