Matsuda N, Kumar N M, Ramakrishnan P R, Lin W L, Genco R J, Cho M I
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo 14214.
Arch Oral Biol. 1993 Jul;38(7):559-69. doi: 10.1016/0003-9969(93)90120-b.
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.
本研究旨在了解表皮生长因子受体（EGF-R）在牙周膜（PDL）成纤维细胞中的作用。培养大鼠PDL成纤维细胞和ROS 17/2.8细胞（高度分化的成骨细胞性骨肉瘤细胞），并用转化生长因子-α（TGF-α）、表皮生长因子（EGF）、地塞米松（Dex）或EGF与Dex的组合进行处理。以对硝基苯磷酸为底物，测定碱性磷酸酶（ALP）活性，其为矿化组织形成细胞的早期分化标志物。对于[125I]-EGF结合的Scatchard分析，将细胞在含有0.2%牛血清白蛋白和0 - 64 ng/ml [125I]-EGF的杜尔贝科改良 Eagle 培养基中于4℃孵育4小时。此外，分别通过免疫沉淀和Northern印迹分析测定EGF-R蛋白的合成和EGF-R mRNA的表达。未处理的PDL成纤维细胞在培养2天时自发ALP活性逐渐增加，从32.4 U/10(6)细胞增加到培养7天时的49.6 U/10(6)细胞。用Dex处理7天后，ALP活性进一步增加至70.8 U/10(6)细胞，而EGF处理则将其降低至19.4 U/10(6)细胞。在Dex和EGF组合存在的情况下培养PDL成纤维细胞，在7天时将Dex诱导的ALP活性从70.8 U降低至41.8 U/10(6)细胞。用TGF-α处理后也发现对ALP活性有类似的抑制作用。相反，ROS细胞在整个培养过程中保持较高的ALP活性（1748 U/10(6)细胞），不受EGF影响。Scatchard分析表明，PDL成纤维细胞具有高亲和力和低亲和力形式的EGF-R，而ROS细胞未检测到任何EGF-R。用Dex处理PDL细胞2天会降低EGF-R蛋白的合成、EGF-R mRNA的表达和EGF-R的数量。相反，EGF处理会增加EGF-R mRNA的表达。这些数据表明，PDL成纤维细胞表达大量的EGF-R，但在Dex的影响下，其在分化为矿化组织形成细胞的过程中数量减少。因此，EGF-R可能在PDL成纤维细胞的表型稳定中发挥作用。
