Abstract
We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken together, IL-1β induced MMP-3-regulated cell proliferation and suppressed apoptosis in odontoblast-like cells derived from iPS and ES cells.
Highlights
Since matrix metalloproteinases (MMPs), a family of calcium and zinc-dependent extracellular matrix (ECM) degrading enzymes, are expressed in physiological and pathophysiological processes, and process virtually any component of the ECM, it has been suggested that MMPs may be important in inflammatory conditions such as rheumatoid arthritis and periodontitis where they participate in inflammation [1,2,3,4]
The odontoblast-like cells derived from induced pluripotent stem (iPS) cells, embryonic stem (ES) cells and KN-3 were cultured in the presence of three concentrations of IL1b (0, 0.25, 2.5 and 25 ng/mL) and MMP-3 induction was assessed using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis
Our data highlights four main points. This is the first report that involved the use of siRNA that targets MMP-3 to elucidate the mechanism on IL-1b-induced proliferation of odontoblast-like cells derived from iPS and ES cells
Summary
Since matrix metalloproteinases (MMPs), a family of calcium and zinc-dependent extracellular matrix (ECM) degrading enzymes, are expressed in physiological and pathophysiological processes, and process virtually any component of the ECM, it has been suggested that MMPs may be important in inflammatory conditions such as rheumatoid arthritis and periodontitis where they participate in inflammation [1,2,3,4]. While it is intuitive that dental pulp destruction may be a function of MMPs, our previous study reported that MMP-3 accelerates wound healing following dental pulp injury. This observation indicates that MMP-3 may be involved in both ECM degradation and the subsequent morphogenesis, wound repair [8,9] and angiogenesis in the inflamed tissue [10,11,12]. Treatment with IL-1b resulted in the potent induction of MMP-3 expression in dental pulp, which contains large numbers of odontoblasts [7] Taken together, these studies suggest that MMP-3 induced by the proinflammatory cytokine IL-1b contributes to the pathophysiology of inflamed dental pulp. We undertook our experiments using purified odontoblast-like cells derived from induced pluripotent stem (iPS) cells [26] and ES cells [27], which are excellent models in which to examine the mechanism of wound healing in vitro
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