Hyperosmotic stress induces cell death in an odontoblast-lineage cell line.
BACKGROUND
Osmotic stress is one of the stimulations related to dental pain caused by caries or dentin hypersensitivity. The mechanism of osmotic-induced dental pain is not completely understood. The purpose of this study was to examine the responses of odontoblasts under sucrose-induced hyperosmotic stress.


METHODS
We used an odontoblast-lineage cell (OLC) line in our experiments. OLCs were stimulated with sucrose to produce hyperosmotic stress. The expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP 1) were detected by using reverse transcriptase polymerase chain reaction assay. The cell viability of OLCs was detected by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The responses accompanied with cell death were detected by using 4-6-diamidino-2-phenylindole staining, Western blotting of caspase-3, and annexin V assay. The expression of mitogen-activated protein kinases (MAPKs) was detected by using Western blot analysis.


RESULTS
DSPP and DMP 1 were not affected by hyperosmotic stress in OLCs. Cell viability decreased over 700 mOsm for 3 hours of cell culture. The shapes of cells and nuclei became irregular and vacuolar under hyperosmotic stress. The expression of cleaved caspase-3 was increased after treatment with hyperosmotic stress. Some propidium iodide-positive cells were detected in flow cytometry analysis. Phosphorylation of 3 MAPKs was induced by hyperosmotic stress. Inhibitors of 3 MAPKs inhibited the hyperosmotic stress-induced decline in cell viability at 500 and 700 mOsm.


CONCLUSIONS
Hyperosmotic stress induces cell death of OLCs with sucrose through a MAPK pathway.
Department of Restorative Dentistry and Endodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
Mari
M
Fujisawa
Masayuki
M
Tokuda
Yoko
Y
Morimoto-Yamashita
Shoko
S
Tatsuyama
Szilvia
S
Arany
Toshihiro
T
Sugiyama
Chiaki
C
Kitamura
Yoshiyuki
Y
Shibukawa
Mitsuo
M
Torii
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D002460:Cell Line; D048430:Cell Shape; D002470:Cell Survival / Q000187:drug effects; D019234:Dentinal Fluid / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D057446:Hydrodynamics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D009804:Odontoblasts / Q000187:drug effects / Q000378:metabolism*; D009997:Osmotic Pressure / Q000502:physiology*; D010750:Phosphoproteins / Q000096:biosynthesis; D010766:Phosphorylation; D012795:Sialoglycoproteins / Q000096:biosynthesis; D013312:Stress, Physiological; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099311:Dentin Sialophosphoprotein
D053148:Caspase 3; D016326:Extracellular Matrix Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D005947:Glucose; D048031:JNK Mitogen-Activated Protein Kinases; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D048051:p38 Mitogen-Activated Protein Kinases; D000099311:Dentin Sialophosphoprotein; C513308:Dmp1 protein, mouse