F-spondin regulates the differentiation of human cementoblast-like (HCEM) cells via BMP7 expression.

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.