Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia.
OBJECTIVE
To analyze the effects of nicotine on palatal fusion inhibition in vitro and determine if nicotine modulated transforming growth factor beta3 or phosphatidylinositol-3 kinase signaling. A second objective was to determine the localization and regulation of nicotinic receptors in the medial edge epithelia (MEE) during palatal fusion.


METHODS
Palatal shelves from embryonic day (E) 13.5 mice were cultured in serum free media and treated with 0, 0.06, 0.6, or 6 mM nicotine, nicotinic receptor antagonist alpha-bungarotoxin, or the combination of nicotine and alpha-bungarotoxin. Tissues harvested at 72 h were analyzed for epithelial-mesenchymal transformation (EMT) and fusion. MEE samples collected at 20 h were analyzed for phosphorylated Akt-Ser473, phosphorylated Smad2, and nicotinic receptors.


RESULTS
Nicotine inhibited palatal fusion in vitro in a dose dependent manner. Activated Akt-Ser473 was greater in control MEE than in nicotine treated tissues; while there was no difference in activated Smad2 between groups. The alpha7 subunit of nicotinic receptor was expressed in MEE during palate fusion and increased in nicotine treated tissues. Alpha-bungarotoxin did not rescue the nicotine treated palates.


CONCLUSIONS
Nicotine treatment had no effect on Smad2, but caused a down regulation of the PI-3 kinase pathway that may have contributed to inhibiting palatal fusion in vitro.
Kang
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Biomedical Sciences, Texas A & M University System, Baylor College of Dentistry, Dallas, TX 75246, USA.
Svoboda
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D000818:Animals; D002038:Bungarotoxins / Q000494:pharmacology; D002972:Cleft Palate / Q000139:chemically induced* / Q000196:embryology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000196:embryology; D007150:Immunohistochemistry; D008648:Mesoderm / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D009538:Nicotine / Q000633:toxicity*; D018722:Nicotinic Agonists / Q000633:toxicity*; D009924:Organ Culture Techniques; D021362:Palate, Hard / Q000187:drug effects* / Q000196:embryology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011978:Receptors, Nicotinic / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053782:Transforming Growth Factor beta3
D002038:Bungarotoxins; D004268:DNA-Binding Proteins; D009538:Nicotine; D018722:Nicotinic Agonists; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011978:Receptors, Nicotinic; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; C497783:Smad2 protein, mouse; C509045:Tgfb3 protein, mouse