Coupling of Ionic Events to Protein Kinase Signaling Cascades upon Activation of α7 Nicotinic Receptor

Abstract

Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of keratinocytes (KCs) during wound epithelialization is one of the major goals in epithelial cell biology. The acetylcholine (ACh)-gated ion channels, or nicotinic ACh receptors (nAChRs), mediate the nicotinergic signaling that controls crawling locomotion of KCs. To elucidate relative contributions of the ionic and protein kinase-mediated events elicited due to activation of ␣7 nAChRs, we quantitated expression of ␣ 2 -integrin gene at the mRNA and protein levels and also measured Rho kinase activity in KCs stimulated with the ␣7 agonist AR-R17779 while blocking the Na ؉ or Ca 2؉ entry and/or inhibiting signaling kinases. The results demonstrated the existence of the two-component signaling systems coupling the ionic events and protein kinase signaling cascades downstream of ␣7 nAChR to simultaneous upregulation of ␣ 2 -integrin expression and activation of Rho kinase. The Raf/MEK1/ERK1/2 cascade up-regulating ␣ 2 -integrin was activated due to both Ca 2؉ -dependent recruitment of Ca 2؉ /calmodulin-dependent protein kinase II and protein kinase C and Ca 2؉ -independent activation of Ras. Likewise the phosphatidylinositol 3-kinase-mediated activation of Rho kinase was elicited due to both Ca 2؉ entry-dependent involvement of Ca 2؉ /calmodulin-dependent protein kinase II and Ca 2؉ -independent activation of Jak2. Thus, although the initial signals emanating from activated ␣7 nAChR are different in nature the pathways intersect at common effector molecules providing for a common end point effect. This novel paradigm of nAChR-mediated coordination of the ionic and metabolic signaling events can allow an auto/paracrine ACh to simultaneously alter gene expression and induce reciprocal changes in the cytoskeleton and contractile system of KCs required to compete a particular step of wound epithelialization.Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of epidermal keratinocytes (KCs) 2 during their lateral migration in a wound bed is one of the major goals in epithelial cell biology. The epithelial and some other types of non-neuronal cells synthesize, degrade, and respond to acetylcholine (ACh) that functions outside the nervous system as an auto/paracrine hormone or a cytotransmitter (for a review, see Ref. 1). The non-neuronal ACh exhibits rapid and profound effects on gene expression due to activation of the muscarinic and nicotinic classes of cholinergic receptors coupling multiple signal transduction pathways. The muscarinic receptors are classic G protein-coupled transmembrane glycoproteins that mediate a metabolic response to ACh through the interactions of G proteins with signal transducing enzymes, leading to increases or decreases of second messengers, ion concentrations, and modulations of protein kinase activities. The nicotinic ACh receptors (nAChRs) are classic representatives of the superfamily of ligand-gated ion channel proteins, or ionotropi...

Molecular mechanisms of synergy of corneal muscarinic and nicotinic acetylcholine receptors in upregulation of E-cadherin expression

“…2C). Consistent with the previously characterized signaling pathway coupling activation of α7 nAChR to upregulated expression of the α 2 -integrin gene in keratinocytes [30], upregulation of E-cadherin in CEC downstream of α7 also included the Ras/Raf/MEK/ERK cascade. In contrast to α2-integrin, the signaling events leading to upregulation of E-cadherin involved both direct and indirect activation of the Ras/Raf/MEK/ERK cascade by α7.…”

“…While the results clearly support a role for M 3 receptors, they do not rule out a role for M 1 and M 5 mAChRs. Activation of α7 nAChR launched the Ras-Raf-MEK-ERK cascade both indirectly, through the Ca 2+ -CaMKII-PKC step, and directly, due to altering the phosporylation status of Ras, which appears to be physically interacting with the α7 receptor protein [30]. Thus, simultaneous stimulation of several subtypes of corneal cholinergic receptors may be required to synchronize and balance ionic and metabolic events in a single cell.…”

“…In contrast to α2-integrin, the signaling events leading to upregulation of E-cadherin involved both direct and indirect activation of the Ras/Raf/MEK/ERK cascade by α7. The former mechanism might result from an agonistdependent confirmation changes of the α7 receptor protein associated with Ras [30], and the latter -due to engagement of the Ca 2+ / CaMKII/PKC cascade, as was evident from inability to completely abolish ing on E-cadherin expression. CEC were transfected with anti-ACh receptor shRNAs or shRNA-NC and incubated for 72 h to achieve maximal silencing of the target receptor gene expression.…”

Nicotinic receptor signaling in nonexcitable epithelial cells: paradigm shifting from ion current to kinase cascade. Focus on “Upregulation of nuclear factor-κB expression by SLURP-1 is mediated by α7-nicotinic acetylcholine receptor and involves both ionic events and activation of protein kinases”

“…Earlier, the authors identified a novel paradigm of nAChR-mediated coordination of the ionic and metabolic signaling events that allows a nicotinic agonist to simultaneously alter gene expression and induce reciprocal changes in the cytoskeleton and contractile system of KCs (13). In the current study, the authors show that a nonionic signaling mechanism mediates upregulation of the NF-B gene expression by SLURP-1 downstream of ␣ 7 -nAChR via coupling of ionic events to protein kinase signaling cascades upon ␣ 7 -nAChR activation.…”

Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes

“…In the first step, the cells were treated at T0, checked every 24 h, and maintained for 144 h (protocol A); in the second step, the cells were treated every 24 h, checked every 24 h, and maintained for 144 h (protocol B). We also tested in protocol A the involvement of muscarinic receptors and calcium using a specific antagonist, treating cells 30 min before stimulation; in particular, for muscarinic receptor 1 we used atropine (7.25 μg/mL; Sigma, Milan, Italy) [Kühne et al, 2015] and for calcium we used H89 (519 ng/mL, inhibitor of PKA kinase; Sigma) and chelerythrine (384 ng/mL, inhibitor of PKC kinase; Cayman Chemicals, Cabru, Milan, Italy) [Chernyavsky et al, 2009;Cappellano et al, 2013].…”