ABSTRACT
Periodontal ligament (PDL) is a thin fibrous connective tissue located between alveolar bone and cementum that remains unmineralized physiologically. It is thus thought that PDL cells possess mechanisms to inhibit mineralization. It has been demonstrated that S100A4, a member of the S100 calcium-binding protein family, is synthesized and secreted by PDL cells, and that it may act as an inhibitor of mineralization. However, the mechanisms of action of S100A4 in mineralization have not been thoroughly clarified. In the present study we investigated the effects of S100A4 inhibition by a short interfering RNA (siRNA) on the expression of osteoblast related genes by human PDL cells. Inhibition of S100A4 by siRNA resulted in increased expression of osteoblastic markers such as osteopontin and osteocalcin, and the osteoblast-specific transcription factors, Runx2/Cbfa1 and Osterix. These results indicate that S100A4 suppresses the expression of osteoblastic genes in PDL cells and may thus inhibit mineralization in the PDL.
Subject(s)
Osteoblasts/metabolism , Osteogenesis/physiology , Periodontal Ligament/metabolism , RNA Interference , S100 Proteins/metabolism , Transcription Factors/metabolism , Cell Differentiation/physiology , Cells, Cultured , Core Binding Factor Alpha 1 Subunit , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Gene Silencing/physiology , Humans , Osteoblasts/cytology , Osteocalcin/physiology , Osteopontin , Periodontal Ligament/cytology , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Sialoglycoproteins/metabolism , Transcription Factor AP-2 , Transcription Factors/genetics , Up-Regulation/physiologyABSTRACT
BACKGROUND: Periodontal ligament fibroblasts (PDLFs) are the cells essential for periodontal regeneration. PDLFs comprise a heterogeneous cell population and consist of several cell subsets that differ in their function. It is known that PDLFs produce osteoblast-related extracellular matrix proteins and show higher alkaline phosphatase (ALP) activity compared with gingival fibroblasts (GFs), implying that PDLFs have osteogenic characterisitics. The aim of the present study was to isolate the osteogenic population of PDLFs according to their expression of ALP. METHODS: PDLFs and gingival fibroblasts were separated into two populations, ALP-positive and ALP-negative, with an immunomagnetic method using a monoclonal antibody against human bone type ALP and magnetic beads conjugated with a secondary antibody. Expression of basic fibroblast growth factor (bFGF) receptor and transforming growth factor (TGF)-beta receptor was investigated in these two populations. Osteoblast-related molecules, osteocalcin, and bone sialoprotein; ALP activity; and effect of bFGF on proliferation were also compared. RESULTS: Effective separation was confirmed in both PDLFs and GFs by flow cytometry. The expression of FGF receptor (FGFR) and TGF-beta receptor was significantly higher in ALP-positive PDLFs than in ALP-negative PDLFs. ALP-positive PDLFs also expressed higher mRNA levels of osteocalcin and bone sialoprotein compared with ALP-negative PDLFs. The mitogenic effect of bFGF on ALP-positive PDLFs was greater than that of ALP-negative PDLFs. CONCLUSIONS: These results indicate that osteoblastic and/or cementoblastic PDLF subsets could be isolated from the PDLF populations using an immunomagnetic method. Magnetic isolation of PDLFs may be a useful tool to obtain the cells which will potentially induce mineralization on the root surface.
