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 α7 nAChRs, we quantitated expression of α₂-integrin gene at the mRNA and protein levels and also measured Rho kinase activity in KCs stimulated with the α7 agonist AR-R17779 while blocking the Na⁺ or Ca²⁺ 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 α7 nAChR to simultaneous up-regulation of α₂-integrin expression and activation of Rho kinase. The Raf/MEK1/ERK1/2 cascade up-regulating α₂-integrin was activated due to both Ca²⁺-dependent recruitment of Ca²⁺/calmodulin-dependent protein kinase II and protein kinase C and Ca²⁺-independent activation of Ras. Likewise the phosphatidylinositol 3-kinase-mediated activation of Rho kinase was elicited due to both Ca²⁺ entry-dependent involvement of Ca²⁺/calmodulin-dependent protein kinase II and Ca²⁺-independent activation of Jak2. Thus, although the initial signals emanating from activated α7 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 ionotropic receptors, mediating the influx of Na⁺ and Ca²⁺ and efflux of K⁺ (2). In neurons, binding of ACh to nAChRs leads to cell membrane depolarization that allows influx of Ca²⁺ through voltage-sensitive calcium channels. Although a high resolution patch clamping technique recorded single channel currents from outside-out patches excised from cultured human epidermal KCs stimulated with ACh, the KCs grown in the medium containing 0.09 mm Ca²⁺ only rarely showed ACh-activated currents (3). This was surprising because under such low Ca²⁺ culture conditions, the nAChR ligands elicit a plethora of biologic effects on KCs (for reviews, see Refs. 4 and 5). The nAChRs regulate survival, proliferation, adhesion, and differentiation of KCs and a large variety of non-neuronal cells and, in particular, play a crucial role in coordinating cellular functions mediating epithelialization of skin (6–8) and lung (9) wounds. Hence elucidation of the signaling events elicited upon agonist binding to keratinocyte nAChRs is crucial for understanding the mechanisms of ACh signaling in non-neuronal cells, which has salient clinical implications.

In non-neuronal cells, nAChRs regulate the expression of many genes. For instance, 118 genes are up-regulated and 97 are down-regulated in the human macrophage-like cell line U937 (10). In KCs, activation of nAChRs alters expression of the genes encoding cell receptor, signal transduction, cell cycle regulation, apoptosis, and cell-cell and cell-substrate adhesion proteins (for reviews, see Refs. 4 and 5). On the keratinocyte plasma membrane, the nicotinergic signals can be elicited due to activation of several classic nAChR subtypes. The homomeric nAChRs expressed in KCs can comprise α7 or α9 subunits, whereas the heteromeric nAChRs can comprise the α3, α5, α9, α10, β1, β2, and β4 subunits, e.g. α3(β2/β4)±α5 and α9α10 (3, 11–15). We have documented that downstream of keratinocyte nAChRs the signaling pathways can involve elevation of intracellular Ca²⁺, activation of the protein kinase C (PKC) isoforms, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), Jak2, phosphatidylinositol 3-kinase (PI3K), Akt, p38 mitogen-activated protein kinase (MAPK), phospholipase C, Src, epidermal growth factor receptor kinase, Rac, and Rho as well as the Ras/Raf-1/MEK/ERK pathway (6–8, 16, 17). There are many potential targets of these signaling cascades, including nuclear processes, metabolic pathways, and structural components of the cytoskeleton, but the receptor-mediated mechanisms of signal induction remain to be identified.

It has been demonstrated that keratinocyte α7 nAChR directs chemotaxis of KCs, which is a critical early step in wound epithelialization, and that the downstream signaling from this receptor in KCs involves intracellular free Ca²⁺, CaMKII, PKC, PI3K, Jak2, and the Ras/Raf-1/MEK/ERK cascade and leads to up-regulation of α₂-integrin gene expression (7, 8, 18). Noteworthy is that PI3K plays a central role in coordinating Rho-mediated signaling events during cell migration (for a review, see Ref. 19). It is well known that α7 subunits form a homomeric receptor/channel that has a high relative permeability to Ca²⁺ (20, 21). Indeed Ca²⁺ ions that enter KCs through α7-containing ACh-gated ion channels can raise the concentration of intracellular free Ca²⁺ in KCs (11, 22), indicating that ionic events contribute to the nicotinergic effects on KCs. However, experiments with several types of non-neuronal cells demonstrated that the nicotinergic effects can be elicited in the absence of Na⁺ or Ca²⁺ entry (23–26). Therefore, the downstream signaling from α7 nAChR expressed in KCs may also proceed via parallel, ionic, and protein kinase signaling pathways.

In this study, we tested a hypothesis that the coordinated regulation of α₂-integrin expression and reciprocal alterations in the cytoskeleton that mediate ACh control of keratinocyte lateral migration through α7 nAChR involves both ionic events and protein kinase signaling cascades. The results demonstrated the existence of the two-component signaling systems allowing simultaneous up-regulation of α₂-integrin expression and ROK activation. The Raf/MEK1/ERK1/2 cascade up-regulating α₂-integrin was activated due to both the Ca²⁺-dependent recruitment of CaMKII and PKC and the Ca²⁺-independent activation of Ras. Likewise the PI3K-mediated activation of ROK was elicited due to both Ca²⁺ entry-dependent involvement of CaMKII and Ca²⁺-independent activation of Jak2. Thus, although the initial signals emanating from activated α7 nAChR are different in nature the pathways intersect at common effector molecules providing for a common end point effect.

MATERIALS AND METHODS

Chemicals and Protein Kinase Assay Kits

The preferential α7 nAChR agonist AR-R17779 (27) was a gift from AstraZeneca Pharmaceuticals (Wilmington, DE). The α7-preferring antagonist methyllycaconitine (MLA) (28) was purchased from Sigma-Aldrich. The noncompetitive inhibitor of the Ras acceptor protein manumycin A (29), the cRaf-1 kinase inhibitor GW5074 (5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone) (30), the Jak2 inhibitor AG-490, and the cell-permeable, potent, and selective inhibitor of MEK “MEK inhibitor I” (31) were from Calbiochem-Novabiochem. The selective inhibitor of CaMKII KN-62, the selective and cell-permeable inhibitor of all PKC isoforms chelerythrine, and the highly specific PI3K inhibitor Ly-294002 were purchased from Axxora, LLC (San Diego, CA). The plasmids encoding the constitutively active MEK1 (CA-MEK) with two point mutations (S218E and S222E) and a deletion of amino acid residues 31–52 and the dominant negative MEK1 mutant (DN-MEK) that contains three point mutations (K97R, S218A, and S222A) and thus could neither be phosphorylated by its activators nor phosphorylate its downstream effector ERKs were purchased from Biomyx Technology (San Diego, CA). The PKC activity assay kit was purchased from Stressgen Bioreagents (Ann Arbor, MI), the phospho-ERK1/2 (Thr-185/Tyr-187) enzyme-linked immunosorbent assay kit was from Millipore (Billerica, MA), the Rho kinase assay kit was from CycLex Co. (Nagano, Japan), and the Jak2 kinase assay kit was purchased from Cell Signaling Technology, Inc. (Boston, MA). All assay kits were used to determine the activity of a corresponding kinase in experimental and control KCs following the protocols provided by the manufacturers.

Keratinocyte Exposure Experiments

Human keratinocyte cultures were started from normal neonatal foreskins (32). The cells were grown in 75-cm² flasks (Corning Glass Works, Corning, NY) in serum-free keratinocyte growth medium containing 5 ng/ml epidermal growth factor and 50 μg/ml bovine pituitary extract (Invitrogen) at 37 °C in a humid, 5% CO₂ incubator at 0.09 mm calcium. The keratinocyte cultures used in experiments were between passages 2 and 4 (∼80% confluent). The cells were washed thrice with oxygenated Krebs-Ringer HEPES buffer, pH 7.4, containing 125 mm NaCl, 4.8 mm KCl, 1.2 mm MgSO₄, 25 mm HEPES, 5.6 mm glucose, and 0.5% bovine serum albumin with or without 2.6 mm CaCl²⁺ and incubated for 3 h at 37 °C with test compound dissolved in Krebs-Ringer HEPES buffer. Then the monolayers were washed with prewarmed keratinocyte growth medium and incubated in this medium for 1 h to recover. After that, the 3-h exposure to test compounds was repeated. Control cells were exposed to Krebs-Ringer HEPES buffer without test compounds. Some experimental cells were incubated in Na⁺-free sucrose buffer in which NaCl was replaced with an isotonic concentration of sucrose (270 mm) and Tris-HCl buffer, pH 7.4, was used instead of HEPES.

Transfection Experiments

The predesigned and tested small interfering RNA (siRNA) targeting human CHRNA7 (GenBank^TM accession number NM_000746) mRNA sequence 5′-GGACAGAUCACUAUUUACA-3′ and negative control siRNA (siRNA-NC) targeting luciferase gene with the target sequence 5′-CGTACGCGGAATACTTCGA-3′ were purchased from Dharmacon (Lafayette, CO). For transfection with siRNAs, we followed the standard protocol described in detail elsewhere (8). Briefly KCs were treated with increasing concentrations of siRNA in the transfection solution with the DharmaFECT^TM 1 siRNA Transfection Reagent (Dharmacon) for 16 h at 37 °C in a humid, 5% CO₂ incubator. On the next day, the transfection medium was replaced by keratinocyte growth medium, and the cells were incubated for 72 h to determine at which time point maximum inhibition of the receptor protein expression occurs. The siRNA transfection efficiency was also assayed using fluorescein isothiocyanate-labeled luciferase GL2 duplex (Dharmacon). It was experimentally established that maximal, i.e. >90%, inhibition of α7 expression occurred 48 h after transfection (data not shown) at which point the cells were used in exposure experiments. The same generic protocol of keratinocyte transfection was used to express MEK1 kinase mutants.

Real Time Quantitative PCR (qPCR) Experiments

Total RNA was extracted from KCs at the end of exposure experiments with the RNeasy Mini kit (Qiagen, Valencia, CA) and used in the qPCR assay of α₂-integrin gene expression as detailed elsewhere (17). The qPCR primers were designed with the assistance of the Primer Express software version 2.0 computer program (Applied Biosystems, Foster City, CA) and the service Assays-on-Demand provided by Applied Biosystems. The qPCRs were performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems) and the TaqMan Universal Master Mix reagent (Applied Biosystems) in accordance with the manufacturer's protocol as described by us in detail elsewhere (33). To correct for minor variations in mRNA extraction and reverse transcription, the gene expression values were normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The data from triplicate samples were analyzed with sequence detector software (Applied Biosystems) and expressed as mean ± S.D. of α₂-integrin mRNA relative to that of control.

In-cell Western Assay

The in-cell Western assay was performed as described by us in detail elsewhere (34) using the reagents and equipment from LI-COR Biotechnology (Lincoln, NE). After exposure of 3 × 10⁴ KCs/well of a 96-well plate to test agents, the experimental and control cells were fixed in situ, washed, permeabilized with Triton solution, incubated with the LI-COR Odyssey Blocking Buffer for 1.5 h, and then treated overnight at 4 °C with a primary rabbit polyclonal antibody to α₂-integrin (Chemicon International, Inc., Temecula, CA). After that, the cells were washed and stained for 1 h at room temperature with a secondary LI-COR IRDye 800CW goat anti-rabbit or IRDye 800CW antibody diluted 1:800. Sapphire700 (1:1000) was used to normalize for cell number/well. The protein expression was quantitated using the LI-COR Odyssey Imaging System.

Statistical Analysis

All experiments were performed in triplicates, and the results were expressed as mean ± S.D. Statistical significance was determined using Student's t test. Differences were deemed significant if the calculated p value was <0.05.

RESULTS

Coupling of the Ionic Events and Protein Kinase Signaling Cascades to Nicotinergic Regulation of α₂-Integrin Gene Expression

We have demonstrated previously that activation of α7 nAChR up-regulates expression of α₂-integrin in human epidermal keratinocytes and that the downstream signaling involves the Ras/Raf-1/MEK1/ERK1/2 cascade (8). To elucidate the relative contribution of the ionic events elicited by influxes of Na⁺ and Ca²⁺ through the ACh-gated homomeric ion channel comprising α7 subunits, we measured by qPCR and in-cell Western assay the changes in expression of the α₂-integrin gene at the mRNA and protein levels, respectively, in KCs stimulated with the α7 nAChR agonist AR-R17779 in the absence of extracellular Na⁺ or Ca²⁺. Under normal culture conditions, AR-R17779 produced multifold increases in the α₂-integrin mRNA and protein that could be abolished in the presence of the α7 antagonist MLA as well as due to transfection with siRNA-α7 (Fig. 1), thus confirming the specificity of the observed effect to α7 nAChR activation. Although elimination of Na⁺ only slightly diminished the AR-R17779 effect (p > 0.05), the AR-R17779-dependent up-regulation of α₂-integrin in Ca²⁺-free medium was inhibited by >50% at three different AR-R17779 concentrations, i.e. 25, 50, and 100 μm (Fig. 1C), suggesting that α7 nAChR-mediated Ca²⁺ influx, rather then cell membrane depolarization, contributes to the signaling cascade leading to up-regulation of the α₂-integrin gene expression. This conjecture was supported by a partial decrease of the AR-R17779-dependent up-regulation of α₂-integrin expression due to inhibition of Ca²⁺ influx through nAChR by Cd²⁺ or Zn²⁺ (35, 36).

FIGURE 1.

Alterations in α₂-integrin gene expression. The qPCR (A) and in-cell Western (B) analyses of the effects of test conditions on the α7 nAChR-dependent activation of α₂-integrin gene expression in human epidermal KCs at the mRNA and protein levels, respectively, were performed as described under “Materials and Methods.” To standardize the analysis, the gene expression ratios in the control cells, i.e. intact KCs, were taken as 1. The following experimental treatments were used: 50 μm AR-R17779, 100 nm MLA, 1 μm MEK inhibitor I (MEK Inh), 10 μm AG-490, 10 μm Ly-294002, 1 μm chelerythrine, 0.3 mm Cd²⁺, 0.1 mm Zn²⁺, and Ca²⁺- or Na⁺-free media. Some cells were transfected with siRNA-α7 versus siRNA-NC and DN-MEK versus CA-MEK. C shows the dose-dependent up-regulation of α₂-integrin in normal (◇) and Ca²⁺-free medium (●). Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n = 3). Asterisks indicate significant (p < 0.05) differences from control KCs. The pound signs indicate significant (p < 0.05) differences from KCs exposed to AR-R17779 alone.

Both in the absence of extracellular Ca²⁺ and in the presence of Cd²⁺ or Zn²⁺, the AR-R17779-dependent elevation of α₂-integrin mRNA and protein was completely blocked by inhibiting MEK1 activity (Fig. 1), indicating that MEK signaling downstream from α7 nAChR can be elicited independently from ion influx. The α7 nAChR pathway coupled to up-regulation of α₂-integrin also involved PKC whose inhibition with chelerythrine produced a reciprocal decrease of the AR-R17779-induced up-regulation of the α₂-integrin gene expression. This putative signaling pathway apparently did not involve Jak2 and PI3K because their inhibition with AG-490 and Ly-294002, respectively, did not alter the AR-R17779-dependent elevation of α₂-integrin mRNA and protein (Fig. 1). These results demonstrated the existence of the two-component signaling system mediating up-regulation of the α₂-integrin gene expression upon stimulation of α7 nAChR with an agonist.

Identification of the Signaling Elements Comprising the Pathways Up-regulating the α₂-Integrin Gene Expression Downstream of α7 nAChR

AR-R17779 produced a dose-dependent increase of ERK1/2 activity, which could be inhibited by MLA and siRNA-α7 (Fig. 2), thus substantiating the notion that downstream signaling from α7 nAChR that up-regulates the α₂-integring gene expression involves the MEK1/ERK1/2 step. Both elimination of the extracellular Ca²⁺ and inhibition of its nAChR permeation through nAChR by Cd²⁺ and Zn²⁺ partially decreased the AR-R17779-dependent ERK activation, whereas elimination of extracellular Na⁺ had no effect. Transfection of KCs with CA-MEK eliminated the inhibitory effects of Ca²⁺ deprivation on both ERK activity (Fig. 2) and α₂-integrin gene expression (Fig. 1). Combining the depravation of extracellular Ca²⁺ with inhibition of MEK in all cases totally blocked AR-R17779-dependent ERK activation. In Ca²⁺-free medium, Ras and Raf inhibitors also totally abolished the AR-R17779 effect, demonstrating that α7 signaling was initiated due to both Ca²⁺ influx and activation of Ras/Raf independently from Ca²⁺ influx.

FIGURE 2.

Measurement of ERK1/2 activity. A, the effects of test conditions on ERK1/2 activity in human epidermal KCs were determined as described under “Materials and Methods” following the protocol for ERK1/2 activity measurement provided by the manufacturer of the assay kit. The ERK1/2 activity in intact KCs was taken as 1. The following experimental treatments were used: 50 μm AR-R17779, 100 nm MLA, 3 μm manumycin A, 0.1 μm GW-5074, 1 μm MEK inhibitor I (MEK Inh), 10 μm AG-490, 10 μm Ly-294002, 1 μm chelerythrine, 0.3 mm Cd²⁺, 0.1 mm Zn²⁺, and Ca²⁺- or Na⁺-free media. Some cells were transfected with siRNA-α7 versus siRNA-NC and DN-MEK versus CA-MEK. B, concentration-dependent effects of AR-R17779 on ERK1/2 activity in normal (◇) and Ca²⁺-free medium (●). Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n = 3). Asterisks indicate significant (p < 0.05) differences from control KCs. The pound signs indicate significant (p < 0.05) differences from KCs exposed to AR-R17779 alone.

To further identify effectors of the nAChR-coupled signaling pathways upstream of the MEK1/ERK1/2 step, we measured the effects of Jak2, PI3K, and PKC inhibitors on the AR-R17779-dependent ERK activation. Only the PKC inhibitor chelerythrine significantly (p < 0.05) decreased ERK activity (Fig. 2), indicating that PKC is involved upstream of the MEK1/ERK1/2 step.

Defining the Mechanism of PKC Involvement in the α7 Signaling Regulating α₂-Integrin Expression

To distinguish between the ionic and protein kinase-mediated mechanisms of PKC engagement in the α7 nAChR signaling up-regulating the α₂-integrin gene expression, we measured PKC activity in KCs deprived of extracellular Ca²⁺ or exposed to kinase inhibitors. The specificity of PKC activation due to agonistic stimulation of α7 nAChR was demonstrated by inhibiting the AR-R17779 effect by MLA and due to transfection with siRNA-α7 but not siRNA-NC (Fig. 3). The AR-R17779-dependent activation of PKC was completely inhibited due to elimination of Ca²⁺ from the culture medium, blocking the nAChR permeability to Ca²⁺ with Cd²⁺ or Zn²⁺, and treatment with the CaMKII inhibitor KN-62 (Fig. 3). The Ras inhibitor manumycin A produced no significant effect (p > 0.05). These results demonstrated that PKC engagement downstream of α7 nAChR activation occurs predominantly due to Ca²⁺ influx and proceeds along the pathway involving CaMKII.

FIGURE 3.

Measurement of PKC activity. The effects of test conditions on PKC activity in human epidermal KCs were measured as described under “Materials and Methods” following the protocol for PKC activity measurement provided by the manufacturer of the assay kit. The PKC activity in control KCs was taken as 1. The cells were exposed to 50 μm AR-R17779, 100 nm MLA, 3 μm manumycin A, 10 μm KN-62, 0.3 mm Cd²⁺, or 0.1 mm Zn²⁺; incubated in Ca²⁺- or Na⁺-free media; or transfected with siRNA-α7 or siRNA-NC. Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n = 3). Asterisks indicate significant (p < 0.05) differences from control KCs. The pound signs indicate significant (p < 0.05) differences from KCs exposed to AR-R17779 alone.

The Signaling Mechanisms Regulating Rho Activity Downstream of α7 nAChR

Because ROK is believed to play a central role in coordinating the cholinergic control of keratinocyte migration through regulation of the integrin gene expression and cytoskeleton rearrangements (5), we next determined whether and how the downstream signaling from α7 nAChR engages ROK. Activation of α7 nAChR with AR-R17779 produced a concentration-dependent increase of the ROK activity that could be inhibited by MLA and transfection with siRNA-α7 (Fig. 4). The AR-R17779-induced ROK activation depended upon Ca²⁺ (Fig. 4B) but not Na⁺ entry. An inhibitor of PI3K completely decreased the AR-R17779-dependent ROK activation; the CaMKII and Jak2 inhibitors decreased it only partially as it remained significantly elevated (p < 0.05), whereas a PKC inhibitor produced no significant effect (p > 0.05) (Fig. 4). Combining Jak2 inhibitor with a blockade of extracellular Ca²⁺ entry or inhibition of CaMKII in all cases completely abolished the AR-R17779-induced activation of ROK. These results indicated that α7 nAChR-mediated activation of ROK may result due to Ca²⁺ influx-dependent engagement of the CaMKII/PI3K step and also independently from Jak2 involvement.

FIGURE 4.

Measurement of ROK activity. A, the effects of test conditions on ROK activity in human epidermal KCs were measured as described under “Materials and Methods” following the protocol for ROK activity measurement provided by the manufacturer of the assay kit. The ROK activity in control KCs was taken as 1. The following experimental treatments were used: 50 μm AR-R17779, 100 nm MLA, 10 μm AG-490, 10 μm Ly-294002, 10 μm KN-62, 1 μm chelerythrine, 0.3 mm Cd²⁺, 0.1 mm Zn²⁺, and Ca²⁺- or Na⁺-free media. Some cells were transfected with siRNA-α7 or siRNA-NC. B, dose-dependent changes in ROK produced by AR-R17779 in normal (◇) and Ca²⁺-free medium (●). Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n = 3). Asterisks indicate significant (p < 0.05) differences from intact control KCs. The pound signs indicate significant (p < 0.05) differences from KCs exposed to AR-R17779 alone.

Activation of Jak2 through α7 nAChR Does Not Depend on Na⁺ and Ca²⁺ Influx

We have demonstrated recently that activation of keratinocyte α7 nAChR leads to recruitment and phosphorylation of Jak2 (18). To determine whether the influx of extracellular Na⁺ and Ca²⁺ through α7 nAChR is necessary for Jak2 activation, we measured Jak2 activity in KCs incubated in a Ca²⁺- or Na⁺-free medium and found no significant changes in the AR-R19779-dependendent Jak2 activation (p > 0.05) (Fig. 5). Inhibition of Ca²⁺ entry through α7 nAChR using Cd²⁺ or Zn²⁺ produced no significant effects either (p > 0.05). Therefore, engagement of Jak2 in the α7 nAChR signaling coupled to activation of ROK does not require Ca²⁺ and Na⁺ influx.

FIGURE 5.

Measurement of Jak2 activity. The effects of test conditions on Jak2 phosphorylation in human epidermal KCs were measured as described under “Materials and Methods” following the protocol for Jak2 activity measurement provided by the manufacturer of the assay kit. The level of Jak2 phosphorylation in control KCs was taken as 1. The cells were exposed to 50 μm AR-R17779, 100 nm MLA, 0.3 mm Cd²⁺, or 0.1 mm Zn²⁺; incubated in Ca²⁺- or Na⁺-free media; or transfected with siRNA-α7 or siRNA-NC. Triplicate experiments were performed with KCs from each of the three cell donors used in this study (n = 3). Asterisks indicate significant (p < 0.05) differences from intact control KCs. The pound signs indicate significant (p < 0.05) differences from KCs exposed to AR-R17779 alone.

DISCUSSION

The obtained results demonstrated for the fist time the synergistic action of the ionic and protein kinase signal transduction events downstream of α7 nAChR in mediating the nicotinergic up-regulation of α₂-integrin expression and ROK activity required for normal keratinocyte migration and wound epithelialization. The α₂-integrin gene expression was up-regulated through the Raf/MEK1/ERK1/2 cascade activated due to both the Ca²⁺-dependent recruitment of CaMKII and PKC and the Ca²⁺-independent activation of Ras (Fig. 6). Likewise the PI3K-mediated activation of ROK was elicited due to both the Ca²⁺ entry-dependent engagement of CaMKII and the Ca²⁺-independent activation of Jak2. Downstream of α7 nAChR, the distinct signals emanated from activated receptor intersected at the common signal transduction effectors, i.e. Raf in the cascade leading to α₂-integrin up-regulation and PI3K in the pathway activating ROK. This novel biologic mechanism may allow an auto/paracrine ACh to synchronize the ionic events and metabolic signaling cascades to simultaneously alter gene expression patterns and induce reciprocal changes in the cytoskeleton and contractile system that altogether drive crawling locomotion of KCs during wound epithelialization.

FIGURE 6.

Cooperation of the ionic and protein kinase signaling elements in the α7 nAChR-dependent pathways up-regulating α₂-integrin and activating ROK. Stimulation of keratinocyte α7 nAChR by its physiologic ligand ACh or the α7-preferring agonist leads to up-regulation of the α₂-integrin gene expression due to activation of the Raf-1/MEK1/ERK1/2 cascade that proceeds via two complementary signaling pathways. One is mediated by the Ca²⁺ entry-dependent CaMKII/PKC activation, and another one is mediated by the Ca²⁺-independent involvement of Ras. At the same time, an agonist stimulation sets off another two-component signal transduction mechanism that leads to PI3K-mediated ROK activation through both ionic and protein kinase-mediated events, such as Ca²⁺-dependent engagement of CaMKII and Ca²⁺-independent activation/autophosphorylation of Jak2 that forms a complex with α7 subunits.

The signaling pathways downstream of nAChRs are involved in regulation of related cellular functions of KCs, such as proliferation, differentiation, apoptosis, adhesion, and migration (for reviews, see Refs. 4 and 5). The diverse nicotinergic signals elicit biologic effects through divergent modifications of a large variety of effector molecules and structural proteins. We have demonstrated previously that activation of α7 nAChR helps orient a keratinocyte toward direction of its future migration via the Ras/Raf/MEK/ERK pathway that up-regulates expression of α₂-integrin required for stabilization of the lamellipodium at the keratinocyte leading edge (8). The α₂-integrin function can switch the Rho-dependent cell shape changes from a resting to a spreading morphology (37). RhoA induces the assembly of contractile actin-myosin filaments (stress fibers) and associated focal adhesion complexes (for a review, see Ref. 38). Thus, the biologic significance of the cooperative signaling from α7 nAChR that simultaneously up-regulates α₂-integrin and activates ROK stems from the fact that both of these effector systems are crucial for initiation of migration of resting KCs at the earliest stage of wound epithelialization.

The AR-R17779-dependent up-regulation of both α₂-integrin expression and ROK activity could be altered by blocking Ca²⁺ entry. This observation is in keeping with our earlier findings that the nAChR-dependent Ca²⁺ influx plays an important role in mediating ACh regulation of keratinocyte migration (22). It is well known that activation of nAChRs elicits Ca²⁺ influx that varies in magnitude between different receptor subtypes and that the ACh-gated ion channels comprising α7 subunits have the greatest Ca²⁺ permeability (20, 39). We have demonstrated previously that Ca²⁺ ions that enter KCs through α7 nAChRs can raise the concentration of intracellular free Ca²⁺ (11, 22). Therefore, it was not surprising that the signaling downstream of α7 nAChR involved CaMKII that, in turn, relayed the downstream signaling to the two separate pathways, one leading to α₂-integrin up-regulation through engagement of PKC and another one activating ROK through PI3K. Indeed it has been established that the Raf/MEK/ERK cascade can be activated independently from Ras in a PKC-dependent manner (40) and that PI3K is positively regulated by Ca²⁺ via CaMKII (41).

In the present study, we demonstrated that in addition to the Ca²⁺-dependent induction of protein kinases the two-component nicotinergic signal transduction systems regulating α₂-integrin gene expression and ROK activation in KCs also include the ion influx-independent events. To prevent Ca²⁺ entry through nAChRs we used Cd²⁺ and Zn²⁺, both of which have been shown to block ACh-evoked currents elicited due to activation of nAChRs (35, 36). Because on the one hand activation of nAChRs in KCs causes a limited depolarization of the cell membrane in the outside-out, but not the whole-cell, patches (3) and on the other hand the voltage-gated Ca²⁺ channels exist in KCs and play a role in skin barrier homeostasis (42), we tested the effect of Na⁺-free medium on AR-R17779 activity. As could be expected based on the fact that in contrast to neurons KCs are generally not considered to be excitable cells, changing the medium did not significantly alter the AR-R17779 effects (p > 0.05).

The functional cooperation of the ionic events and protein kinase signaling cascades was illustrated by the fact that inhibition of either Ca²⁺ entry or protein kinase activities produced only partial inhibition of the activity of AR-R17779, whereas a combined inhibition of both ionic and protein kinase pathways completely abolished its effects. Because others also reported that inhibition of Ca²⁺ influx using Ca²⁺ entry blockers or a Na⁺-free incubation solution only partially blocks the nicotinergic effects (23, 25, 43–45), a similarly arranged two-component signaling system apparently operates in other cells downstream of nAChRs.

In both models of keratinocyte deprivation of the extracellular Ca²⁺ entry, i.e. due to treatment with Cd²⁺ or Zn² and using a Ca²⁺- or Na⁺-free medium, AR-R17779 activated Ras and Jak2. The exact nature of the non-excitable events associated with AR-R17779 binding to α7 nAChR is not fully understood because nAChR subunits do not have intrinsic tyrosine kinase activity (46). Perhaps the AR-R17779 signaling observed by us in the absence of Ca²⁺ entry was elicited due to formation of functional multiprotein complexes of α7 subunit with the signaling proteins anchored to the cell membrane, such as Ras and Jak2 (47, 48). We have reported previously that activation of keratinocyte α7 nAChR leads to phosphorylation and activation of Jak2 (16, 18) and also initiates Ras-mediated signaling (8, 18, 49). Currently there is no published evidence that α7 can form complexes with Ras, but such a paradigm has been established for Jak2. It has been shown that agonist-induced complex formation between α7 and Jak2 leads to subsequent activation of PI3K (50) in agreement with the results of the present study. Our observations are also consistent with the notion that an important role of the Jak2/PI3K signaling cascade is the physiologic control of cell migration (51).

The demonstration of the functional coupling of nAChRs to signaling kinases further unveils a novel function of the subunit proteins that mediate nicotinergic effects in non-excitable cells. As already mentioned, Jak2 physically associates with α7 upon stimulation with an agonist and becomes phosphorylated and activated (52). The α7 subunit can also physically and functionally associate with Src family kinases (53–56), whereas the subunits α3-α5 and β2 exhibit a positive interaction with the G protein subunits G_(o)α and Gβγ (57). The nAChR proteins can associate with both protein kinase and protein-tyrosine phosphatase in large multimeric complexes (58). For instance, it was proposed that Src-associated phosphatase functions in early signaling events emanating from nAChR and that the Src to phosphatase ratio determines the functional state of associated nAChR (54). Our own data indicate strongly that constant stimulation of keratinocyte α7 nAChR by autocrine/paracrine ACh controls Src activity and that interruption of ACh signaling releases Src activity, leading to catenin phosphorylation and weakening cell cohesion (59). Of particular interest is an observation that nicotinergic up-regulation of mitogenesis involves an α7-mediated recruitment of β-arrestin and thus facilitates Src activation (60). Therefore, the mode of interaction of nAChRs with the signal transduction molecules may vary depending on the type of receptor subunit involved, the kind of signaling pathway involved in mediating a particular nicotinergic function, and the cell type responding to an agonist. Thus, elucidation of the mechanisms of α7 nAChR signaling in non-excitable cells like KCs opens a door for a novel approach to pharmacologic regulation of the cellular functions regulated through the nAChRs expressed in non-neuronal cells.