Yamada S, Ozaki N, Tsushima K, Yamaba S, Fujihara C, Awata T, Sakashita H, Kajikawa T, Kitagaki J, Yamashita M, Yanagita M, Murakami S
Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
Department of Periodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan
J Dent Res. 2016 Aug;95(9):1026-33. doi: 10.1177/0022034516645796. Epub 2016 Apr 29.
Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.
牙周韧带（PDLs）在牙槽骨和牙骨质的重塑过程中发挥着重要作用。牙周组织转录组的特征仍不完整，对PDL特征的深入了解有助于开发新的再生疗法。在此，我们旨在生成并分析一个大型人类PDL转录组。我们从正畸治疗患者身上获取PDL，分离RNA，并使用载体加帽法从20000多个克隆中构建互补DNA文库。我们的结果显示，58%的序列是全长的。此外，我们的分析表明，表达频率最高的基因包括I型胶原蛋白、III型胶原蛋白和蛋白酶的基因。我们还发现了5个以前未在人类PDL中报道过表达的基因。为了确定哪些高表达基因可能对PDL细胞分化很重要，我们使用实时聚合酶链反应来测量它们在分化细胞中的表达。在所测试的基因中，半胱氨酸蛋白酶组织蛋白酶K的上调幅度最大，因此我们测量了它在几种组织以及已知高表达组织蛋白酶K的破骨细胞中的相对表达。我们的结果显示，PDL细胞表达组织蛋白酶K的水平与破骨细胞相似，两者的表达水平均高于其他测试组织。我们还测量了细胞分化过程中组织蛋白酶K的蛋白质表达和酶活性，发现两者在此过程中均增加。免疫细胞化学实验表明，组织蛋白酶K定位于溶酶体内部。最后，我们研究了在细胞分化过程中抑制组织蛋白酶K的作用，发现抑制组织蛋白酶K会刺激钙化结节形成，并增加I型胶原蛋白和骨钙素基因的表达水平。基于这些结果，组织蛋白酶K似乎在人类PDL细胞分化为硬组织形成细胞的过程中调节胶原纤维的积累。
