Interleukin-1β-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro
Interleukin-1β-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro
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DOI：
10.1007/pl00005822
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发表时间：
1999-05-01
影响因子：
4.2
通讯作者：
Cho, MI
中科院分区：
文献类型：
--
作者：
Chien, HH;Lin, WL;Cho, MI
通过形态学分析、免疫沉淀和北方印迹分析等方法，探讨白细胞介素1 β（IL-1 β）抑制牙周膜（PDL）细胞体外矿化结节形成的机制。PDL细胞从牙槽中2天大的凝块中获得，并在含有10%胎骨血清（FBS）和抗生素的Dulbecco改良Eagle培养基（DMEM）中培养。融合细胞在抗坏血酸（AA）、β-甘油磷酸盐（GP）和地塞米松（Dex）或IL-1存在下生长长达3周。在GP和AA存在下培养的PDL细胞不分化，但Dex、GP和AA（Dex组）处理的PDL细胞发生分化，显示出不同形态特征的四个阶段（汇合、多层、结节和矿化）。而IL-1、Dex、GP和AA（IL-1组）处理的细胞形成了多层膜，但未形成矿化结节。电子显微镜检查表明，右旋糖酐诱导的矿化结节含有多层成纤维细胞，许多胶原纤维，以及致密的球状和融合的电子致密补丁，与许多磷灰石晶体。IL-1组的结节样结构也由多层成纤维细胞组成，但它们仅含有少量胶原纤维，没有致密的球状或融合的斑块。Von Kossa染色证实Dex组中存在大量矿化结节，而IL-1组中矿化结节较少。然而，IL-1处理的细胞的北方印迹分析揭示了I型胶原（Col I）、分泌蛋白、酸性和富含半胱氨酸（Cys）、骨桥蛋白（OPN）、碱性磷酸酶（ALP）、骨唾液酸蛋白（BSP）和骨钙素（OC）的mRNA的存在，其表达模式和水平与Dex组相当。[S-35]-甲硫氨酸标记后，细胞/基质层和培养基中OPN和BSP的免疫沉淀分析显示，Dex组的OPN和BSP主要沉积在矿化结节中，而IL-1组的OPN和BSP则释放到培养基中。免疫胶体金标记显示OPN和BSP在Dex组矿化结节中的定位，但在IL-1组的结节样结构中没有明显的标记。有趣的是，与Dex组相比，IL-1治疗使胶原酶mRNA的表达增加了7倍。这些数据表明，IL-1诱导的PDL细胞形成的未矿化结节的结果，而不是从下调形成的基质蛋白，这在矿化过程中起着至关重要的作用，从他们释放到培养基中。最后，由IL-1上调的胶原酶合成可能参与该过程。
The mechanism by which interleukin-1 beta (IL-I) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-I group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture media after [S-35]-methionine labeling showed their deposition primarily in the mineralized nodules of the Dex group, and their release into the media in the IL-1 group. Immunogold labeling demonstrated the location of OPN and BSP in mineralized nodules of the Dex group, but no significant labeling occurred in the nodule-like structures from the IL-I group. Interestingly, IL-1 treatment increased the expression of collagenase mRNA by sevenfold, compared with that of the Dex group. These data suggest that the IL-1-induced formation of unmineralized nodules by PDL cells results not so much from the downregulated formation of matrix proteins, which plays a crucial role in the mineralization process, as from their release into the culture media. Finally, collagenase synthesis upregulated by IL-1 may be involved in this process.