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Epidermal growth factor receptor transactivation is required for mitogen-activated protein kinase activation by muscarinic acetylcholine receptors in HaCaT keratinocytes.

Ockenga W, Kühne S, Bocksberger S, Banning A, Tikkanen R - Int J Mol Sci (2014)

Bottom Line: Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling.The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells.The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany. Wymke.Ockenga@biochemie.med.uni-giessen.de.

ABSTRACT
Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.

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Inhibition of epidermal growth factor receptor (EGFR) kinase activity prevents cholinergic activation of Akt and extracellular signal regulated kinase (ERK). (A) Serum-starved HaCaT cells were stimulated with 1 mM CCh, 100 µM nicotine or 16 nM EGF for 30 min. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted. The results are representative of three independent experiments; (B) To inhibit EGFR kinase activity, starved HaCaT cells were pretreated with the EGFR kinase inhibitor PD 153035 or a non-inhibiting analogue AG9 (both 1 µM) for 5 min. Control cells here represent the AG9 treated cells. The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitor or its analogue. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; (C,D) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated control. Bars represent the mean ± SD of four independent experiments. Statistical analysis was performed with two-way ANOVA; **p < 0.01, ***p < 0.001; (E–H) Transcriptional response upon EGF or CCh stimulation (30 min–4 h) was measured at protein (E, Egr1 expression) or mRNA level (measured with quantitative real-time PCR) for Egr1 (F), cFos (G), and Dusp1 (H). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed with one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (E) Shows a representative result of three independent experiments.
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ijms-15-21433-f001: Inhibition of epidermal growth factor receptor (EGFR) kinase activity prevents cholinergic activation of Akt and extracellular signal regulated kinase (ERK). (A) Serum-starved HaCaT cells were stimulated with 1 mM CCh, 100 µM nicotine or 16 nM EGF for 30 min. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted. The results are representative of three independent experiments; (B) To inhibit EGFR kinase activity, starved HaCaT cells were pretreated with the EGFR kinase inhibitor PD 153035 or a non-inhibiting analogue AG9 (both 1 µM) for 5 min. Control cells here represent the AG9 treated cells. The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitor or its analogue. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; (C,D) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated control. Bars represent the mean ± SD of four independent experiments. Statistical analysis was performed with two-way ANOVA; **p < 0.01, ***p < 0.001; (E–H) Transcriptional response upon EGF or CCh stimulation (30 min–4 h) was measured at protein (E, Egr1 expression) or mRNA level (measured with quantitative real-time PCR) for Egr1 (F), cFos (G), and Dusp1 (H). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed with one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (E) Shows a representative result of three independent experiments.

Mentions: To study if a cholinergic stimulus leads to activation of ERK and Akt, HaCaT cells were stimulated for 30 min with the cholinergic agonist carbachol (CCh, 1 mM), EGF (16 nM) or nicotine (100 µM), and the activation of Akt and ERK was analyzed with phospho-specific antibodies (Figure 1A). Whereas stimulation with CCh and EGF resulted in a robust activation of both Akt and ERK, nicotine did not induce any activation of ERK and only a minimal change in Akt phosphorylation. Thus, the activation of ERK and Akt in HaCaT cells by cholinergic stimuli is likely to be mediated by the muscarinic receptors.


Epidermal growth factor receptor transactivation is required for mitogen-activated protein kinase activation by muscarinic acetylcholine receptors in HaCaT keratinocytes.

Ockenga W, Kühne S, Bocksberger S, Banning A, Tikkanen R - Int J Mol Sci (2014)

Inhibition of epidermal growth factor receptor (EGFR) kinase activity prevents cholinergic activation of Akt and extracellular signal regulated kinase (ERK). (A) Serum-starved HaCaT cells were stimulated with 1 mM CCh, 100 µM nicotine or 16 nM EGF for 30 min. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted. The results are representative of three independent experiments; (B) To inhibit EGFR kinase activity, starved HaCaT cells were pretreated with the EGFR kinase inhibitor PD 153035 or a non-inhibiting analogue AG9 (both 1 µM) for 5 min. Control cells here represent the AG9 treated cells. The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitor or its analogue. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; (C,D) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated control. Bars represent the mean ± SD of four independent experiments. Statistical analysis was performed with two-way ANOVA; **p < 0.01, ***p < 0.001; (E–H) Transcriptional response upon EGF or CCh stimulation (30 min–4 h) was measured at protein (E, Egr1 expression) or mRNA level (measured with quantitative real-time PCR) for Egr1 (F), cFos (G), and Dusp1 (H). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed with one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (E) Shows a representative result of three independent experiments.
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getmorefigures.php?uid=PMC4264234&req=5

ijms-15-21433-f001: Inhibition of epidermal growth factor receptor (EGFR) kinase activity prevents cholinergic activation of Akt and extracellular signal regulated kinase (ERK). (A) Serum-starved HaCaT cells were stimulated with 1 mM CCh, 100 µM nicotine or 16 nM EGF for 30 min. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted. The results are representative of three independent experiments; (B) To inhibit EGFR kinase activity, starved HaCaT cells were pretreated with the EGFR kinase inhibitor PD 153035 or a non-inhibiting analogue AG9 (both 1 µM) for 5 min. Control cells here represent the AG9 treated cells. The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitor or its analogue. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; (C,D) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated control. Bars represent the mean ± SD of four independent experiments. Statistical analysis was performed with two-way ANOVA; **p < 0.01, ***p < 0.001; (E–H) Transcriptional response upon EGF or CCh stimulation (30 min–4 h) was measured at protein (E, Egr1 expression) or mRNA level (measured with quantitative real-time PCR) for Egr1 (F), cFos (G), and Dusp1 (H). Bars represent the mean ± SD of three independent experiments. Statistical analysis was performed with one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001. (E) Shows a representative result of three independent experiments.
Mentions: To study if a cholinergic stimulus leads to activation of ERK and Akt, HaCaT cells were stimulated for 30 min with the cholinergic agonist carbachol (CCh, 1 mM), EGF (16 nM) or nicotine (100 µM), and the activation of Akt and ERK was analyzed with phospho-specific antibodies (Figure 1A). Whereas stimulation with CCh and EGF resulted in a robust activation of both Akt and ERK, nicotine did not induce any activation of ERK and only a minimal change in Akt phosphorylation. Thus, the activation of ERK and Akt in HaCaT cells by cholinergic stimuli is likely to be mediated by the muscarinic receptors.

Bottom Line: Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling.The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells.The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany. Wymke.Ockenga@biochemie.med.uni-giessen.de.

ABSTRACT
Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.

Show MeSH
Related in: MedlinePlus