Effects of long noncoding RNA H19 on cementoblast differentiation, mineralisation, and proliferation.

OBJECTIVE: Cementum which is a layer of thin and bone-like mineralised tissue covering tooth root surface is deposited and mineralised by cementoblasts. Recent studies suggested long noncoding RNA H19 (H19) promotes osteoblast differentiation and matrix mineralisation, however, the effect of H19 on cementoblasts remains unknown. This study aimed to clarify the regulatory effects of H19 on cementoblast differentiation, mineralisation, and proliferation. MATERIAL AND METHODS: An immortalised murine cementoblast cell line OCCM-30 was used in this study. H19 expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) during OCCM-30 cell differentiation. OCCM-30 cells were transfected with lentivirus or siRNA to up-regulate or down-regulate H19, then the levels of runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), osteocalcin (Bglap) were tested by RT-qPCR or western blot. Alizarin red staining, ALP activity assay and MTS assay were performed to determine the mineralisation and proliferation ability of OCCM-30 cells. RESULTS: H19 was dramatically increased during OCCM-30 cell differentiation. Overexpression of H19 increased the levels of Runx2, Sp7, Alpl, Ibsp, and Bglap and enhanced ALP activity and the formation of mineral nodules. While down-regulation of H19 suppressed the above cementoblast differentiation genes and inhibited ALP activity and mineral nodule formation. However, the proliferation of OCCM-30 cells was not affected. CONCLUSIONS: H19 promotes the differentiation and mineralisation of cementoblasts without affecting cell proliferation.

miR-146a-5p attenuates IL-1ß-induced IL-6 and IL-1ß expression in a cementoblast-derived cell line.

OBJECTIVE: miR-146a is widely induced during the immune response. However, little is known about the biogenesis, function and mechanism of miR-146a in cementoblasts during the pathogenesis of periodontitis. This study aimed to investigate the effects of miR-146a in murine cementoblast-derived OCCM-30 cells following IL-1ß stimulation. MATERIALS AND METHODS: OCCM-30 cells were cultured and exposed to IL-1ß. IL-6, IL-1ß and TNF-α, and miR-146a-5p expression was assessed by qRT-PCR. Mimics/inhibitors were transiently transfected into cells to determine the function of miR-146a-5p. Signalling pathways including p38 MAPK, ERK1/2 and NF-κB were studied by using specific inhibitors. The indicated proteins were measured by Western blot analysis and ELISA. RESULTS: In IL-1ß-stimulated OCCM-30 cells, the expression levels of miR-146a-5p along with IL-6 and IL-1ß increased in a time-dependent manner. The ERK1/2, p38 MAPK and NF-κB pathway were activated upon IL-1ß stimulation. Blocking the NF-κB pathway decreased IL-6, IL-1ß and miR-146a-5p expression. The overexpression of miR-146a-5p reduced IL-6 and IL-1ß expression, while the inhibition of miR-146a-5p increased IL-6 and IL-1ß expression in IL-1ß-treated OCCM-30 cells. miR-146a-5p attenuated IL-6 and IL-1ß expression via the IRAK1/TRAF6 pathway. CONCLUSION: This study suggested that miR-146a-5p attenuates IL-1ß-induced inflammatory factors in cementoblast-derived cell line.

Irisin promotes cementoblast differentiation via p38 MAPK pathway.

OBJECTIVE: Irisin is a newly identified exercise-induced myokine which can affect glucose metabolism and cortical bone mass and strength. However, the influence of irisin on cementoblasts remains largely unknown. MATERIAL AND METHODS: An immortalized mouse cementoblast cell line OCCM-30 was used in this study. Cementoblast differentiation markers and PGC-1α in cells cultured with mineral induction medium were evaluated by qRT-PCR. Cementoblast mineralization was evaluated by alizarin red staining. Differentiation markers and the activity of p38 MAPK pathway under irisin stimulation were assessed by qRT-PCR or Western blot analysis. p38 MAPK pathway inhibitor SB203580 or p38 siRNA was used to further identify the regulatory mechanism. Cell proliferation treated with irisin was examined by CCK-8 method. RESULTS: The expression of Runx2, osterix, ALP, and PGC-1α was up-regulated consistently under mineral induction. The formation of mineralized nodules was increased by irisin. Runx2, osterix, ALP, and osteocalcin were obviously up-regulated under irisin stimulation as well as the activity of p38 MAPK pathway. When pretreated with SB203580 or p38 siRNA before irisin stimulation, the irisin-induced differentiation was distinctly suppressed. OCCM-30 cell proliferation was enhanced when treated with high-dose irisin for long time. CONCLUSION: Irisin can promote the differentiation of cementoblasts via p38 MAPK pathway.

Effects of IL-1ß on MMP-9 Expression in Cementoblast-Derived Cell Line and MMP-Mediated Degradation of Type I Collagen.

It has been reported that matrix metalloproteinases (MMPs) are induced by many cytokines, and they are involved in various inflammatory processes, including periodontitis. However, the effects of interleukin-1ß (IL-1ß) on MMP-9 expression in cementoblasts, the cells responsible for cementum production, remain largely unknown. In this study, we used qPCR and gelatin zymogram analysis to show that IL-1ß upregulated MMP-9 expression in cementoblast-derived cell line. Several signaling pathways, such as ERK1/2, JNK, p38, and AP-1 (c-Fos and ATF-2), were activated in response to IL-1ß stimulation. Furthermore, enhancement of AP-1 activity by IL-1ß was further confirmed by the AP-1 reporter assay and the electrophoretic mobility shift assay (EMSA). Pretreatment with specific inhibitors of ERK1/2 (U0126), JNK (SP600125), and AP-1 (tanshinone IIA) attenuated IL-1ß-induced MMP-9 expression. In addition, inhibitors of ERK1/2 (U0126) and JNK (SP600125) attenuated IL-1ß-enhanced AP-1 activity. This suggested that IL-1ß stimulated AP-1 activation, at least partially, through ERK1/2 and JNK signaling pathways. Moreover, we found that IL-1ß also upregulated the expression of MMP-13 and enhanced MMP-mediated degradation of type I collagen. Collectively, these results suggested that IL-1ß induced MMP-9 expression by activation of AP-1 through the ERK1/2 and JNK signaling pathways in cementoblast-derived cell line and enhanced MMP-mediated collagen degradation possibly by MMP-13 and MMP-9.

Inhibition of Stat3 signaling pathway decreases TNF-α-induced autophagy in cementoblasts.

Autophagy is a self-digestive process that eliminates impaired or aged proteins and potentially toxic intracellular components to maintain homeostasis. We previously demonstrated that TNF-α played a critical role in cementoblast differentiation, mineralization and apoptosis; however, the effect of TNF-α on cementoblast autophagy has remained unclear. In this study, an elevated immunofluorescence signal of LC3B and autophagic vacuoles, autophagosomes and autolysosomes were detected under TNF-α stimulation in OCCM-30 cells. Autophagy-related genes and proteins, Beclin-1, LC3A and Atg-5, were significantly upregulated by TNF-α in a time- and concentration-dependent manner. During this process, the activity of Stat3 was dramatically enhanced and when the activity of Stat3 was blocked by either a specific chemical inhibitor or siRNA transfection before TNF-α stimulation, the TNF-α-induced upregulation of autophagy-related genes and proteins was strongly inhibited. Our results suggest that TNF-α induced autophagy in cementoblasts was dependent, or partially dependent on the activity of Stat3 signaling pathway.