Receptor-mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium influx and intracellular concentration.

Early stages of wound healing rely on the ability of keratinocytes (KCs) to move over the denuded dermis to re-epithelialize the defect. The agarose gel keratinocyte outgrowth system (AGKOS) is an in vitro model of skin re-epithelialization designed to study the migratory function of KCs. Endogenously secreted acetylcholine controls crawling locomotion of KCs in AGKOS by binding to the cholinergic receptors of both the nicotinic and the muscarinic classes that are expressed by KCs. In this study, we used AGKOS to elucidate the nicotinic pathway of cholinergic control of keratinocyte migration. Activation of the nicotinic acetylcholine receptors decreased the migration distance of KC in a dose-dependent fashion without altering cell viability. Nicotine also increased in a dose-dependent manner transmembrane influx of (45)Ca(2+), and caused a transient rise in the concentration of [Ca(2+)](i). Perfect correlation between concentration responses found in the migration and (45)Ca(2+) influx assays suggested that nicotine-induced inhibition of crawling locomotion relies on modulation of Ca(2+) metabolism in KCs. The effects of nicotine could be mediated by the alpha3- and the alpha7-containing nicotinic receptors visualized on KCs by immunostaining. Long-term incubation with nicotine up-regulated alpha7 and down-regulated alpha3 expression. Thus, nicotine exerts inhibitory effects on keratinocyte migration, and Ca(2+) serves as a second messenger in the signaling pathway. These results help explain deleterious effects of nicotine on wound re-epithelialization, and suggest that smoking may delay wound healing via nicotinic receptor-mediated pathway.

The pathophysiological significance of nondesmoglein targets of pemphigus autoimmunity. Development of antibodies against keratinocyte cholinergic receptors in patients with pemphigus vulgaris and pemphigus foliaceus.

OBJECTIVES: To determine whether nondesmoglein (non-Dsg) autoantibodies are pathogenic and whether they recognize keratinocyte cholinergic receptors that control cell adhesion because antikeratinocyte autoimmunity in patients with pemphigus vulgaris is not limited to the development of autoantibodies to Dsg. DESIGN: To determine whether non-DSg autoantibodies are pathogenic, we sought to induce pemphigus in genetically engineered neonatal mice lacking Dsg 3 using pemphigus vulgaris IgGs that did not cross-react with Dsg 1. To determine whether pemphigus autoimmunity involves keratinocyte cholinergic receptors, the latter were separated from cell membranes of human keratinocytes, tagged with the covalent label [3H]propylbenzilylcholine mustard, and used as an antigen in a radioimmunoprecipitation assay of 34 pemphigus vulgaris and 6 pemphigus foliaceus serum samples. SETTING: The dermatologic clinics of the University of Minnesota, Minneapolis; the Mayo Clinic, Rochester, Minn; and the University of California-Davis Medical Center, Sacramento. PATIENTS: Serum samples were collected from 34 patients with pemphigus vulgaris and 6 patients with pemphigus foliaceus (aged 31-89 years) and from 7 age-similar patients of both sexes with nonpemphigus blistering or the following immune-mediated conditions: pemphigoid gestation, bullous drug eruption, lupus erythematosus, erythema nodosum, urticaria, acute contact dermatitis, and skin ulcers. MAIN OUTCOME MEASURES: Clinical, laboratory, and histopathologic findings. RESULTS: Extensive skin blistering accompanied by the Nikolsky sign and suprabasilar acantholysis was induced in the Dsg3null mice that received pemphigus, but not normal human IgGs. In the radioimmunoprecipitation assays for reactivity with cholinergic receptors, the mean radioactivity precipitated by pemphigus serum samples significantly exceeded both normal- and disease-control levels (P = .001-.02). The mean individual levels of radioactivity precipitated by 34 pemphigus vulgaris and pemphigus foliaceus serum samples (85%) exceeded control values by a mean of approximately 2.6 times. CONCLUSIONS: Autoantibodies to keratinocyte cell-surface molecules other than Dsg 1 and Dsg 3 can induce clinical features of pemphigus vulgaris. Patients with pemphigus vulgaris and those with pemphigus foliaceus develop IgG antibodies that precipitate radiolabeled cholinergic receptors. Because these receptors control keratinocyte adhesion and motility, their inactivation by autoantibodies may elicit intracellular signals that cause disassembly of desmosomes, leading to acantholysis and blistering.

Analysis of transcripts derived from sequences upstream of the bidirectional mouse thymidylate synthase promoter.

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element and is capable of directing transcriptional initiation with approximately equal strength and over broad initiation windows in both directions. The goal of the present study was to determine if the TS promoter directs the transcription of a second gene that is upstream of the TS gene by characterizing the transcripts that correspond to the upstream sequences. RNA blot analyses revealed the presence of 1.4 and 5 kb cytoplasmic, polyadenylated transcripts that include sequences upstream of the TS promoter. The transcripts were much more abundant in a cell line in which the TS gene is amplified. S1 nuclease protection assays showed that the transcripts have multiple 5' termini. An exon trap approach identified a potential splice donor site that might correspond to the 3' end of the first exon of the upstream gene. A cDNA library was probed with a sequence from the putative first exon, and six different cDNA clones were isolated. However, analysis of the sequences of the cDNAs revealed that the upstream transcripts were not spliced at the potential 3' donor site but instead extended into a repetitive LINE (long interspersed nuclear element) sequence that begins 0.3 kb upstream of the TS promoter. RNase protection assays confirmed that the in vivo transcripts extend into the LINE element. Therefore it appears that the upstream transcripts are unlikely to correspond to a functional mRNA molecule.

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation.

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human beta-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless beta-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes.

Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.