Center of Oral Clinical Examination, Hiroshima University Hospital, Hiroshima University, Hiroshima 734-8553, Japan.
Biochem Biophys Res Commun. 2012 Feb 10;418(2):229-33. doi: 10.1016/j.bbrc.2011.12.155. Epub 2012 Jan 8.
Cementum plays an important role in the attachment of connective tissue to the root surface. However, the detailed mechanism of cementum formation has not yet been clarified. We previously established human cementoblast-like cell lines (HCEM) and human periodontal ligament cell lines (HPL) by infection of hTERT gene. Using those cell lines, we compared the gene expression of them and identified F-spondin as a cementoblast specific gene. In this study, to clarify the role of F-spondin in the differentiation of periodontal ligament cells to cementoblasts, we compared the gene expression of F-spondin-overexpressed HPL (HPL-spondin) cells with HPL parent cells. We found that several genes expressed higher level in HPL-spondin cells than in HPL cells, such as heparin sulfate 6-sulfotranferase, calcitonin-related polypeptide beta, bone morphogenetic proteins 7 (BMP7), BMP2 and BMP8B. Among those genes, we focused on BMP7 and examined the interaction between F-spondin and BMP7, because BMP7 was reported to enhance cementoblast function. Moreover, we further examined the effect of BMP7 peptide on the expression of mineralization-associated genes in HCEM cells. RT-PCR and real-time PCR analyses showed that HPL-spondin expressed BMP7, but not HPL cells. And BMP7 and phospho-Smad1/5/8 protein production were detected in HPL-spondin by Western blot. siSPON1 inhibited expression of type I collagen, runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP) mRNA in HCEM cells. And the mineralization tended to be decreased in siSPON1 treated cells by ALZ staining and the quantification analysis. Moreover, we examined the effect of BMP7 peptide on the gene expressions of HCEM cells by RT-PCR. Increase of the osteopontin and BSP mRNA was observed in BMP7 treated HCEM cells. These findings indicate that F-spondin regulates the differentiation of HCEM cells via BMP7 expression.
牙骨质在连接组织与牙根表面的附着中起着重要作用。然而，牙骨质形成的详细机制尚未阐明。我们之前通过感染 hTERT 基因建立了人牙周膜成纤维细胞系（HPL）和人牙周膜成牙骨质细胞系（HCEM）。利用这些细胞系，我们比较了它们的基因表达，并鉴定出 F-spondin 为成牙骨质细胞特异性基因。在这项研究中，为了阐明 F-spondin 在牙周膜细胞向成牙骨质细胞分化中的作用，我们比较了过表达 F-spondin 的 HPL（HPL-spondin）细胞与 HPL 亲本细胞的基因表达。我们发现，HPL-spondin 细胞中几种基因的表达水平高于 HPL 细胞，例如肝素硫酸 6-硫酸转移酶、降钙素相关多肽β、骨形态发生蛋白 7（BMP7）、BMP2 和 BMP8B。在这些基因中，我们重点研究了 BMP7，并研究了 F-spondin 与 BMP7 之间的相互作用，因为 BMP7 被报道能增强成牙骨质细胞的功能。此外，我们进一步研究了 BMP7 肽对 HCEM 细胞矿化相关基因表达的影响。RT-PCR 和实时 PCR 分析表明，HPL-spondin 表达 BMP7，但 HPL 细胞不表达。通过 Western blot 检测到 HPL-spondin 中 BMP7 和磷酸化 Smad1/5/8 蛋白的产生。siSPON1 抑制 HCEM 细胞中 I 型胶原、runt 相关转录因子 2（RUNX2）和骨涎蛋白（BSP）mRNA 的表达。通过 ALZ 染色和定量分析，siSPON1 处理的细胞矿化趋于减少。此外，我们通过 RT-PCR 研究了 BMP7 肽对 HCEM 细胞基因表达的影响。在 BMP7 处理的 HCEM 细胞中观察到骨桥蛋白和 BSP mRNA 的增加。这些发现表明 F-spondin 通过 BMP7 表达调节 HCEM 细胞的分化。
