Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro.
This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.
Matsuda
N
N
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo 14214.
Kumar
N M
NM
Ramakrishnan
P R
PR
Lin
W L
WL
Genco
R J
RJ
Cho
M I
MI
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002454:Cell Differentiation; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression; D012516:Osteosarcoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D010513:Periodontal Ligament / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D004815:Epidermal Growth Factor; D003907:Dexamethasone; D066246:ErbB Receptors; D000469:Alkaline Phosphatase