Disease-associated extracellular matrix suppresses osteoblastic differentiation of human periodontal ligament cells via MMP-1

Abstract

Fibronectin (FN) fragments found in chronic inflammatory diseases, including periodontal disease and arthritis, may contribute to tissue destruction in part via induction of matrix metalloproteinases (MMPs). We previously showed that the 120-kDa FN fragment containing the central cell binding domain (120FN) dose dependently induces MMP-1 (collagenase-1) in human periodontal ligament (PDL) cells, whereas intact FN did not elicit this response. Recently, we found that an increase in MMP-1 expression is accompanied by a decreased osteoblastic phenotype in PDL cells. We hypothesized that 120FN inhibits osteoblastic differentiation of PDL cells by inducing MMP-1. Effects of increasing concentrations of 120FN on MMP-1 expression and on osteoblastic markers were assessed in cultured PDL cells using Western blotting, qRT-PCR, and collagen degradation and alkaline phosphatase (AP) activity assays. The 120FN dose dependently increased MMP-1 expression and activity, concomitant with a decrease in AP activity. The increase in collagenase activity was largely attributed to increased MMP-1 expression. Concurrent with the decrease in AP activity, the 120FN reduced baseline and dexamethasone-induced gene expression of specific osteoblastic markers, Runx2 and osteonectin, and diminished mineralized nodule formation. Finally, siRNA inhibition of 120FN-induced MMP-1 reduced collagenase expression and rescued the AP phenotype to baseline levels. These findings suggest that disease-associated 120FN, in addition to having direct effects on tissue destruction by upregulating MMPs, could contribute to disease progression by impeding osteoblastic differentiation of osteogenic PDL cells and, consequently, diminish bone regeneration.

Figure 1

Purification scheme for the 120FN fragment. Human plasma FN was digested with a-chymotrypsin (A) and the 120 FN fragment containing the central cell binding-domain was collected in successive fractions (1 to 6) in flow through from gelatin-Sepharose (B) and heparin-Sepharose (C) column chromatography. The appropriate fractions were further purified by dialysis (D). For steps A to D, the samples were subjected to 10% SDS PAGE gel electrophoresis and stained by Coomassie blue. The identity of the purified fragment was further confirmed by Western blotting using an anti-central cell binding-domain (CCBD) antibody (E).

Figure 2

The 120FN dose-dependently induces MMP-1 in PDL cells. Early passage PDL cells were cultured in serum-free medium in the absence or presence of 120FN (0.001–0.1 μM) for 3 days. Substrate zymography on cell-conditioned media demonstrated a dose-dependent increases in a 53/58 gelatinolytic proteinase (53/58G) (A), which was identified as MMP-1 and its dose-dependent induction confirmed on Western blot (B). Extracted RNA was assayed by qRT-PCR for MMP-1 gene expression (C), cell-conditioned media was assayed for collagenase activity by FITC-collagen degradation assay (D), and cell lysates were assayed for AP activity (E). Data are shown as mean (+ SD) fold change relative to control baseline levels (Ct) for three experiments performed in triplicate. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3

The 120FN induces collagenase activity and concomitantly decreases AP activity in PDL cells. Early passage PDL cells were cultured in serum-free medium in the absence or presence of 120FN (0.1 μM) for various time points. Cell-conditioned medium assayed by substrate zymography demonstrated a time-dependent 120FN-mediated induction of 53/58 gelatinolytic proteinase (53/58G) (A), identified as MMP-1 by Western blotting (B). This was accompanied by a time-dependent induction of MMP-1 gene expression assayed by qRT-PCR (C) and collagenase activity analyzed by FITC-collagen degradation assay on cell-conditioned medium (D). Assays on cell extract revealed that relative to control untreated cells, 120FN inhibits the time-dependent increase in AP activity (E). Data are shown as mean (+ SD) fold change relative to control baseline levels (Ct) for three experiments performed in triplicate. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 4

The 120FN inhibits the expression of osteoblastic markers in PDL cells concomitant to induction of MMP-1. PDL cells were cultured in serum-free medium in the absence or presence of 0.1μM 120FN. Cells cultured with the pro-osteogenic dexamethasone (Dex) were used as positive controls. Cell conditioned medium was assayed for collagenase activity (A), cell lysates analyzed for AP activity (C), and total RNA was extracted and subjected to qRT-PCR for MMP-1 (B), Runx2 (D) and ON (E). Data are shown as mean (+ SD) fold change relative to control (Ct) for three independent experiments, each performed in triplicate. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 5

The inhibition of AP phenotype by the 120FN is mediated through its induction of MMP-1. Early passage PDL cells were transfected with 100 pmole of siRNA to MMP-1 (siMMP-1) or scrambled control siRNA (siC) and maintained in serum-free medium for 3days. The cell-conditioned medium was assayed by Western blot for MMP-1 (A) and collagenase activity (B), and the cell extracts assayed for AP activity (C). Data are shown as mean (+ SD) fold change relative to control for three independent experiments performed in triplicate. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 6

The 120FN inhibits mineralized nodule formation in PDL cells. PDL cells were cultured in osteogenic/mineralizing FN-free serum medium without or with 120FN (0.1 μM) for 14 days. The cells and matrix were subjected to von Kossa staining (A). For quantification, mineralized nodules were counted for a total of six different fields per well (B). Data are shown as mean (+ SD) fold change relative to control baseline levels for three experiments performed in triplicate (***, p < 0.001).