ABSTRACT
Dental fluorosis is caused by chronic high-level fluoride (F(-)) exposure during enamel development, and fluorosed enamel has a higher than normal protein content. Matrix metalloproteinase 20 cleaves enamel matrix proteins during the secretory stage, and KLK4 further cleaves these proteins during the maturation stage so that the proteins can be reabsorbed from the hardening enamel. We show that transforming growth factor ß1 (TGF-ß1) can induce Klk4 expression, and we examine the effect of F(-) on TGF-ß1 and KLK4 expression. We found that in vivo F(-) inhibits Klk4 but not Mmp20 transcript levels. LacZ-C57BL/6-Klk4 (+/LacZ) mice have LacZ inserted in frame at the Klk4 translation initiation site so that the endogenous Klk4 promoter drives LacZ expression in the same temporal/spatial way as it does for Klk4. KLK4 protein levels in rat enamel and ß-galactosidase staining in LacZ-C57BL/6-Klk4 (+/LacZ) mouse enamel were both significantly reduced by F(-) treatment. Since TGF-ß1 induces KLK4 expression, we tested and found that F(-) significantly reduced Tgf-ß1 transcript levels in rat enamel organ. These data suggest that F(-)-mediated downregulation of TGF-ß1 expression contributes to reduced KLK4 protein levels in fluorosed enamel and provides an explanation for why fluorosed enamel has a higher than normal protein content.
Subject(s)
Cariostatic Agents/pharmacology , Dental Enamel Proteins/drug effects , Fluorides/pharmacology , Kallikreins/antagonists & inhibitors , Transforming Growth Factor beta1/drug effects , Ameloblasts/drug effects , Ameloblasts/pathology , Amelogenin/analysis , Amelogenin/drug effects , Animals , Cell Line, Tumor , Cells, Cultured , Down-Regulation , Enamel Organ/drug effects , Gene Knock-In Techniques , Kallikreins/analysis , Lac Operon/drug effects , Matrix Metalloproteinase 20/drug effects , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Rats , Rats, Sprague-Dawley , beta-Galactosidase/analysisABSTRACT
Fluorosed enamel can be porous, mottled, discolored, hypomineralized, and protein-rich if the enamel matrix is not completely removed. Proteolytic processing by matrix metalloproteinase-20 (MMP20) and kallikrein-4 (KLK4) is critical for enamel formation, and homozygous mutation of either protease results in hypomineralized, protein-rich enamel. Herein, we demonstrate that the lysosomal proteinase cathepsin K is expressed in the enamel organ in a developmentally defined manner that suggests a role for cathepsin K in degrading re-absorbed enamel matrix proteins. We therefore asked if fluoride directly inhibits the activity of MMP20, KLK4, dipeptidyl peptidase I (DPPI) (an in vitro activator of KLK4), or cathepsin K. Enzyme kinetics were studied with quenched fluorescent peptides with purified enzyme in the presence of 0-10 mM NaF, and data were fit to Michaelis-Menten curves. Increasing concentrations of known inhibitors showed decreases in enzyme activity. However, concentrations of up to 10 mM NaF had no effect on KLK4, MMP20, DPPI, or cathepsin K activity. Our results show that fluoride does not directly inhibit enamel proteolytic activity.
Subject(s)
Dental Enamel Proteins/drug effects , Dental Enamel/enzymology , Fluorides/pharmacology , Peptide Hydrolases/drug effects , Ameloblasts/drug effects , Amelogenesis/drug effects , Amelogenesis/physiology , Animals , Cathepsin C/analysis , Cathepsin C/drug effects , Cathepsin K/antagonists & inhibitors , Cathepsin K/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/administration & dosage , Dipeptides/pharmacology , Dose-Response Relationship, Drug , Enamel Organ/drug effects , Enzyme Inhibitors/pharmacology , Kallikreins/antagonists & inhibitors , Kallikreins/drug effects , Leucine/analogs & derivatives , Leucine/pharmacology , Matrix Metalloproteinase 20/drug effects , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/administration & dosage , Protease Inhibitors/pharmacology , Serine Proteinase Inhibitors/administration & dosage , Serine Proteinase Inhibitors/pharmacology , Sulfones/administration & dosage , Sulfones/pharmacology , Swine , Time FactorsABSTRACT
The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.