Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

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.

Blockade of epidermal growth factor and its receptor and axial elongation in experimental myopia

“…It is interesting that some of the molecules that affect axial elongation, for example, the m1‐muscarinic acetylcholine receptor, can transactivate EGFR 36,37 . Studies have shown that EGFR, as a single transmembrane receptor tyrosine kinase, can become catalytically activated and dimerize through a m1 mAChR‐regulated pathway that requires protein kinase C but is independent of EGF itself.…”

Cholinergic Transactivation of the EGFR in HaCaT Keratinocytes Stimulates a Flotillin-1 Dependent MAPK-Mediated Transcriptional Response

“…Transactivation of the EGFR, which is induced by MMP/ADAM mediated proteolytic release of the ligands of the EGFR, is required for ERK activation upon cholinergic stimulus in HaCaT cells [ 28 ]. We here tested how cholinergic stimulation of HaCaT cells affects the expression of selected EGFR ligands and MMPs/ADAMs by measuring their transcript levels.…”

“…Our earlier findings have shown that a metalloproteinase that can be inhibited by the broad spectrum MMP/ADAM inhibitor, batimastat, mediates the release of EGF-like ligands during cholinergic EGFR transactivation [ 28 ]. We thus focused our analysis on MMPs/ADAMs that are inhibited by batimastat and/or have been implicated in the release of the EGF-like ligands studied above.…”

“…In addition to the cholinergic regulation of keratinocyte physiology, EGFR and its ligands also play an important regulatory role in the epidermis [ 26 ]. We have earlier shown that in the human keratinocyte-like cell line HaCaT [ 27 ], cholinergic stimulation results in transactivation of the EGFR, which then induces a downstream response through the MAPK cascade [ 28 ]. In HaCaT cells, cholinergic MAPK activation is fully dependent on EGFR transactivation by mAChRs, mainly M 3 and M 1 , but it does not involve Src kinases, protein kinase C or phosphatidylinositol 3-kinase.…”

“…In HaCaT cells, cholinergic MAPK activation is fully dependent on EGFR transactivation by mAChRs, mainly M 3 and M 1 , but it does not involve Src kinases, protein kinase C or phosphatidylinositol 3-kinase. However, metalloproteinases that can be inhibited by the broad spectrum inhibitor, batimastat, are required for the EGFR ligand release [ 28 ].…”