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First published online 19 October 2004
doi: 10.1242/jcs.01492

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Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

¹ Department of Dermatology, University of California Davis, 4860 Y Street, Sacramento, CA 95817, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (72K): [in a new window]   Fig. 1. Schemes of the AGKOS plates used for studying cholinergic effects on KC migration. (A) Chemokinesis AGKOS assay. (B) Chemotaxis AGKOS assay.

View larger version (41K): [in a new window]   Fig. 2. Cholinergic effects on KC random migration. (A) Effects of cholinergic agonists on random migration of human KCs. Second-passage foreskin KCs were loaded into the chemokinesis AGKOS plates, incubated overnight, to allow cells to settle, after which the increasing concentrations of the agonists nicotine (Nic), epibatidine (Epi) and choline (Cln) diluted in KGM vs KGM alone (control) were added. The agonist concentrations are shown on the ordinate axis as 10–x M. The plates were incubated for 10 days with daily refreshing of the culture medium. The random migration distance (RMD) was measured in µm (Fig. 1A). Triplicate experiments were performed with KCs from each cell donor and the combined results were averaged. An asterisk denotes statistical significance (P<0.05) compared with control, taken as 100%. (B) Different effects of cholinergic antagonists on random migration of human KCs. Second-passage foreskin KCs were loaded into the chemokinesis AGKOS plates and incubated in KGM (baseline migration distance) or in KGM containing test agents. The medium containing test agents was replaced every day during the 10-day course of the migration assay. Experimental KCs were exposed to 20 µM hemicolinium-3 (HC-3), 1 mM carbachol (CCh), a combination of HC-3 and CCh, 50 µM mecamylamine (Mec), 50 µM tubocurarine (Tub), 1 µM -bungarotoxin (BTX), 100 µM -conotoxin MII (CtxMII), 1 µM -conotoxin AuIB (CtxAuIB) or 5 µM strychnine (Str). The results are expressed as means±s.d. of nontreated control, taken as 100%. Asterisks indicate significant (P<0.05) differences from control. Significant differences between specific experimental conditions are indicated in the graph with arrows at the top. (C) Effects of functional inhibition of nAChR expression on random migration of human KCs. Representative results of western-blot analysis of the effect of antisense oligonucleotides (AsOs) vs control oligonucleotides (COs) on the expression of 3, 5, 7, 9, ß2 or ß4 nAChR subunits in human KCs. Cells were seeded in 24-well plates at a density of 5x104 per well and incubated overnight to allow cell adherence to the dish bottom. KCs were then transfected with COs and AsOs (Table 1). The receptor bands appeared at the expected molecular weights. (D) Alterations in KC random migration because of nAChR subunit gene silencing. Second-passage human KCs were loaded into AGKOS plates, incubated for 18 hours to allow cells to settle and transfected with COs or anti-nAChR-subunit AsOs and incubated for 10 days in KGM containing 20 µM HC-3 and 1 mM CCh, with daily changes of culture medium. Functional inactivation of 3, ß2 and ß4 significantly (P<0.05) decreased RMD compared with the values determined in the control cultures that were exposed to HC-3 and CCh without transfection with control or experimental AsOs (taken as 100%). The results are expressed as means±s.d. of control, taken as 100%. Significant differences are indicated with asterisks and arrows at the top. (E) Effects of nAChR gene knockout on random migration of murine KCs. Second-passage KCs grown from the epidermis of at least three neonatal 3–/–, 5–/–, 7–/–, 9–/–, ß2–/–, ß4–/– or their +/+ littermates were loaded into AGKOS plates, exposed to a combination of 20 µM HC-3 and 1 mM CCh, and incubated for 10 days, after which migration was stopped and RMD values were measured. The results are means±s.d. of RMD of wild-type KC littermates, taken as 100%. Asterisks indicate significant (P<0.05) differences from control values in each subgroup. The lack of 3, ß2 and ß4 significantly (P<0.05) decreased RMD, whereas lack of 7 significantly (P<0.05) increased RMD. (F) Effects of the nAChR signaling modifiers on random migration of human KCs. The migration assay of human KCs was performed in the chemokinesis AGKOS plates as described for A. The ACh synthesis was inhibited with 20 µM HC-3 and the cells were stimulated to migrate with 1 mM CCh in the absence (control) or presence (experiment) of 10 µM BAPTA/AM, 10 µM KN-62 or KN-93, 1 µM chelerythrine (Chlrn), 1 µM Gö-6976, 5 µM rottlerin (Rtln), 100 pg ml–1 toxin B (TxB), 10 µg ml–1 C3 exoenzyme (C3), 5 µM Y-27632, 100 nM wortmannin (Wtmn) or 10 µM Ly-294002. The results are expressed as means±s.d.% of the control cultures that were not exposed to signal modifiers, taken as 100%. Asterisks indicate significant (P<0.05) differences from control. Significant differences between specific experimental conditions are indicated in the graph by arrows at the top.

View larger version (31K): [in a new window]   Fig. 3. Cholinergic effects on KC directional migration. (A) Chemotactic effects of cholinergic agonists on human KCs. Second-passage foreskin KCs were loaded into the chemotaxis AGKOS plates and incubated overnight to allow cells to settle, after which increasing concentrations of the agonists nicotine (Nic), epibatidine (Epi) and choline (Cln) diluted in PBS vs PBS alone (control; C) were added to the chemoattractant well (Fig. 1B). The agonist concentrations are shown on the ordinate axis as 10–x M. The plates were incubated for 10 days with daily refreshment of the chemoattractant solution. A statistically significant (P<0.05) increase in the directional migration distance (DMD) was observed starting at 10 pM nicotine, 1 pM epibatidine and 0.1 mM choline. The DMD of control and experimental cells are expressed as means±s.d. µm. An asterisk denotes statistical significance, P<0.05, compared with control. (B) Subtype-selective antagonists of nAChRs exhibit differential inhibitory effects on directional migration of KCs. The chemotaxis of human KCs was elicited using the most efficient concentration of each agonist shown in A. The antagonists 50 µM mecamylamine (Mec), 1 µM -bungarotoxin (BTX) and 5 µM strychnine (Str) were added directly to the KC well (Fig. 1B), being dissolved in KGM that was changed daily. The DMD values are expressed as means±s.d. µm. An asterisk denotes statistical significance (P<0.05) compared with DMD of the control KCs that were not exposed to antagonists. Significant differences between DMD values of KCs treated with Mec vs BTX are indicated by arrows at the top. (C) Decreased directional migration of human KCs with silenced 7 nAChR. Second-passage human KCs were loaded into the chemotaxis AGKOS plates, incubated for 18 hours to allow cells to adhere to the dish bottom and transfected with COs or anti-nAChR subunit AsOs, after which 1 mM choline was added to the chemoattractant well (Fig. 1B) and the incubation was continued for 10 days. The results are expressed as means±s.d. of control. (D) Decreased directional migration of murine KCs from 7 knockout mice. Second-passage KCs grown from the epidermis of at least three neonatal 3–/–, 5–/–, 7–/–, 9–/–, ß2–/– or ß4–/– mice, or their +/+ littermates were loaded into the chemotaxis AGKOS plates and allowed to migrate towards the concentration gradient of choline for 10 days, as described in C. An asterisk denotes statistical significance (P<0.05) compared with the DMD of 7+/+ KCs. (E) Effects of the nAChR signaling modifiers on directional migration of human KCs. The chemokinetic response to choline was measured in the chemotaxis AGKOS plates using HC-3 (20 µM) treated KCs in which endogenous ACh was substituted by exogenously added CCh (1 mM) (as in Fig. 2F). The cells were fed with KGM containing 10 µM BAPTA/AM, 10 µM KN-62 or KN-93, 1 µM chelerythrine (Chlrn), 1 µM Gö-6976, 5 µM rottlerin (Rtln), 100 pg ml–1 toxin B (TxB), 10 µg ml–1 C3 exoenzyme (C3), 5 µM Y-27632, 100 nM wortmannin (Wtmn) or 10 µM Ly-294002. The results are expressed as means±s.d. of untreated control, taken as 100%. Asterisks indicate significant (P<0.05) differences from control.

View larger version (79K): [in a new window]   Fig. 4. Immunolocalization of 3 and 7 nAChRs in chemotaxing KCs. Colonies of second-passage human foreskin KCs grown on coverslips inserted underneath the agar gel of the chemotaxis AGKOS plates (Fig. 1B) were fixed to avoid cell-membrane permeabilization. (A) Dual immunolabeling with antibody to nAChR subunit and ß1 integrin analysed by deconvolution microscopy. The images show cell-membrane colocalization (yellow) of 3 or 7 nAChR subunit (green) with the integrin ß1 (red). The images of nAChR subunits and ß1 integrin were acquired in a single plane. Bar, 30 µm. (B) Immunostaining with the 3- or 7-specific rabbit antibodies before and 45 minutes and 90 minutes after addition of 1 mM choline to the chemoattractant well. The vertical arrow indicates the position of the chemoattractant well. Notice that the haphazard pattern of receptor distribution on the cell membrane of intact KC changes after exposure to a chemoattractant. The 7 receptor accumulated at the leading edge (lamellipodium), decorating the filopodia (anterior cytoplasmic spikes). The 3 receptor immunoreactivity was abundant at the frontal cell area behind the leading edge. Specific staining was eliminated when the primary rabbit anti-receptor antibody was omitted or when the rabbit antiserum was preincubated with the peptide used for immunization. No immunostaining was observed when the KCs were treated with pre-immune sera obtained from the same rabbits (data not shown). Bar, 25 µm.

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