RESUMEN
Since their introduction more than 50 years ago, water flossers (also known as oral irrigators) have been subject to numerous studies on their safety, efficacy, and delivery of antimicrobial agents, particularly in relation to their ability to impact clinical parameters such as plaque, bleeding, gingivitis, pocket depth, and calculus. Evidence based on such studies and decades of use by the public continues to support their safety and efficacy and disproves suggestions of possible detrimental effects on the attachment, junctional epithelium, or pocket depth.
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Dispositivos para el Autocuidado Bucal , Higiene Bucal/instrumentación , Humanos , AguaRESUMEN
Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca(2+) influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca(2+) permeability, such as alpha5-containing alpha3 nAChR and alpha7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the alpha5 and alpha7 expression in KCs, which could be abolished by mecamylamine and alpha-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: alpha3(beta2/beta4) > alpha3(beta2/beta4)alpha5 > alpha7 > alpha7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the alpha3(beta2/beta4) to alpha3(beta2/beta4)alpha5 nAChR transition predominantly involved protein kinase C, alpha3(beta2/beta4)alpha5 to alpha7 nAChR transition-Ca(2+)/calmodulin-dependent protein kinase II and p38 MAPK, and alpha7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca(2+) permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products.
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Queratinocitos/fisiología , Proteínas del Tejido Nervioso/fisiología , Nicotina/farmacología , Receptores Nicotínicos/fisiología , Fumar/fisiopatología , Contaminación por Humo de Tabaco/efectos adversos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Bungarotoxinas/farmacología , Butadienos/farmacología , Carbazoles/farmacología , Conotoxinas/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Factor de Transcripción GATA2/fisiología , Humanos , Imidazoles/farmacología , Indoles/farmacología , Queratinocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Nitrilos/farmacología , Fenoles/farmacología , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Piridinas/farmacología , ARN Interferente Pequeño/farmacología , Receptores Nicotínicos/biosíntesis , Transducción de Señal , Tirfostinos/farmacología , Regulación hacia Arriba , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
To gain a mechanistic insight into nicotinic receptor-dependent morbidity of tobacco products in the oral cavity, we studied effects of exposures of normal human oral keratinocytes (KCs) for 24 h to environmental tobacco smoke (ETS) vs. equivalent concentration of pure nicotine. The exposed KCs showed a multifold increase of nuclear factor-kappaB (NF-kappaB) at the mRNA and protein levels, which could be significantly (p<0.05) diminished by alpha-bungarotoxin or transfection with anti-alpha7 small interfering RNA. An increased protein-binding activity of NF-kappaB also could be prevented by blocking alpha7 signaling. The use of pathway inhibitors demonstrated that the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent upregulation of NF-kappaB. Thus, exposure of KCs to tobacco may lead to the pathobiologic effects via an intracellular signaling pathway downstream of alpha7 that proceeds through the Ras/Raf-1/MEK1/ERK steps leading to upregulated expression and transactivation of NF-kappaB.
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Queratinocitos/efectos de los fármacos , FN-kappa B/metabolismo , Nicotiana/química , Nicotina/farmacología , Receptores Nicotínicos/fisiología , Bungarotoxinas/farmacología , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo , FN-kappa B/genética , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Receptores Nicotínicos/genética , Transducción de Señal/efectos de los fármacos , Humo , Transfección , Receptor Nicotínico de Acetilcolina alfa 7 , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly alpha7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into alpha7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of alpha7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the alpha7 antagonist alpha-bungarotoxin (alphaBTX) or transfected with anti-alpha7 small interfering RNA (siRNA-alpha7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent up-regulation of STAT-3. Targeted mutation of the alpha7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by alphaBTX or siRNA-alpha7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from alpha7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral alpha7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.
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Queratinocitos/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nicotiana/toxicidad , Receptores Nicotínicos/metabolismo , Factor de Transcripción STAT3/genética , Animales , Células Cultivadas , Humanos , Janus Quinasa 2 , MAP Quinasa Quinasa 1/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Boca , Nicotina/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Factor de Transcripción STAT3/efectos de los fármacos , Transfección , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Receptor Nicotínico de Acetilcolina alfa 7 , Proteínas ras/metabolismoRESUMEN
The biologic role of novel cholinergic toxin-like signaling peptides termed SLURP (secreted mammalian Ly-6/uPAR-related protein) in the mucocutaneous epithelium is a subject of intense research. Previous studies demonstrated that SLURP-1 activates the alpha7 subtype of keratinocyte nicotinic acetylcholine receptors (nAChRs) and facilitates keratinization and programmed cell death, and that the level of SLURP-2 was found to be upregulated several fold in the hyperproliferative skin of patients with psoriasis. In this study, we demonstrated for the first time that human epidermal and oral keratinocytes secrete SLURP-2. We cloned human SLURP-2 and produced the mouse monoclonal antibody 341F10-1F12 that visualized SLURP-2 in the cytoplasm of normal human epidermal and oral keratinocytes grown in culture. In epidermis, SLURP-2 was found predominantly in the suprabasal compartment, whereas in the attached gingiva-in the lowermost epithelial layers. Recombinant SLURP-2 (rSLURP-2) competed with nicotinic radioligands for binding to keratinocytes, showing a higher affinity to the [3H]epibatidine- than [3H]nicotine-labeled sites. Treatment with rSLURP-2 significantly (P < 0.05) increased the number of keratinocytes in culture and their resistance to apoptosis, which could be abolished by mecamylamine more efficiently than alpha-bungarotoxin. By real-time PCR and in-cell western, rSLURP-2 significantly (P < 0.05) downregulated gene expression of the differentiation markers loricrin, filaggrin, and cytokeratins 1 and 10, and pro-apoptotic Bax, Bad, and caspase 3 which were elevated by high extracellular calcium, and rSLURP-2 also abolished activation of caspases 3 and 8 caused by camptothecin. These results indicated that SLURP-2 competes with acetylcholine predominantly at the alpha3 nAChR, and that receptor ligation with SLURP-2 delays keratinocyte differentiation and prevents apoptosis. Thus, the different effects observed for SLURP-1 and -2 can be explained by their differential binding to the nAChR subtypes expressed in keratinocytes. These findings present a novel paradigm of the physiologic regulation of mucocutaneous epithelial cells by locally produced small hormone-like peptide molecules, and open novel directions toward better understanding and treating of skin and mucosal diseases.
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Queratinocitos/química , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/fisiología , Transducción de Señal/fisiología , Piel/química , Acetilcolina/análisis , Acetilcolina/fisiología , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Epiteliales/química , Células Epiteliales/fisiología , Proteínas Filagrina , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI , Encía/química , Encía/citología , Humanos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Membrana Mucosa/química , Membrana Mucosa/citología , Unión Proteica , Receptores Nicotínicos/análisis , Receptores Nicotínicos/fisiología , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Fenómenos Fisiológicos de la Piel , Regulación hacia Arriba/fisiologíaRESUMEN
Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-alpha3 small interfering RNA or treated with the alpha3beta2-preferring antagonist alpha-conotoxin MII. Functional inactivation of alpha3-mediated signaling in alpha3-/- mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal alpha3+/+ and alpha3-/- littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified alpha3beta2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by alpha3beta2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-kappaB, and STAT-1. The expression of the nAChR subunits alpha5 and beta2 and the muscarinic receptor subtypes M(2) and M(3) was also altered. This novel mechanism offers innovative solutions to ameliorate the tobacco-related cell damage and intercede in disease pathways, and may shed light on general mechanisms regulating and driving tobacco-related morbidity in human cells.