Correction: Han et al. MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells. Int. J. Mol. Sci. 2023, 24, 7433.

In the original publication, there was a mistake in Figure 4J,K as published [...].

MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro.

Tissue-resident macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) are found in multiple tissues and organs. We aimed to evaluate the dynamics and biological functions of LYVE-1+ macrophages in dental pulp during post-injury tissue remodeling. Immunofluorescence staining of mouse embryos revealed that LYVE-1+ macrophages colonized dental pulp before birth. In mature rat molar dental pulp, LYVE-1+ macrophages were the main subset of macrophages expressing CD163, an M2 marker, and were distributed throughout the tissue. In response to dental pulp injury induced by cavity preparation, LYVE-1+ macrophages quickly disappeared from the affected area of the pulp and gradually repopulated during the wound healing process. RAW264.7 mouse macrophages cultured with a mixture of macrophage colony-stimulating factor, interleukin-4, and dexamethasone increased LYVE-1 expression, whereas lipopolysaccharide-stimulation decreased LYVE-1 expression. Enforced expression of Lyve1 in RAW264.7 cells resulted in increased mRNA expression of matrix metalloproteinase 2 (Mmp2), Mmp9, and vascular endothelial growth factor A (Vegfa). Lyve1-expressing macrophages promoted the migration and tube formation of human umbilical vein endothelial cells. In conclusion, LYVE-1+ tissue-resident M2-like macrophages in dental pulp showed dynamism in response to pulp injury, and possibly play an important role in angiogenesis during wound healing and tissue remodeling.

Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/ß-catenin transcriptional cofactor BCL9.

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/ß-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/ß-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and ß-catenin expression and BCL9-ß-catenin co-localization. In addition, BCL9 formed a complex with ß-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/ß-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/ß-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

HIF1α inhibits LPS-mediated induction of IL-6 synthesis via SOCS3-dependent CEBPß suppression in human dental pulp cells.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPß), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPß.

Anti-inflammatory roles of microRNA 21 in lipopolysaccharide-stimulated human dental pulp cells.

microRNAs are small noncoding RNA molecules that regulate RNA silencing and posttranscriptional gene expression, and many microRNAs are involved in inflammatory processes. In particular, microRNA 21 (miR-21) is upregulated in inflammatory environment and reported to induce anti-inflammatory responses. However, the involvement of miR-21 in pulpal inflammation and the precise mechanisms of anti-inflammatory reactions induced by miR-21 remain unclear. We hypothesized that miR-21-5p expression is induced in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs) and that miR-21-5p downregulates the proinflammatory cytokine expression in LPS-stimulated hDPCs. We found that miR-21-5p was upregulated in LPS-stimulated hDPCs concomitant with elevated proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) phosphorylation. miR-21-5p and cytokine expression were downregulated by BAY11-7085 and caffeic acid phenylethyl ester (CAPE), specific and potent NF-κB inhibitors. Enforced expression of miR-21-5p downregulated the Toll-like receptor (TLR)/NF-κB signaling via reducing the expression of TNF receptor-associated factor 6 (TRAF6) and programmed cell death 4 (PDCD4), which further induced the decrease of proinflammatory cytokine expression. hDPCs forcibly overexpressing miR-21-5p downregulated the LPS-induced expression of TNF receptor-associated factor 6 (TRAF6; a component of the Toll-like receptor [TLR]/NF-κB signaling pathway), programmed cell death 4 (PDCD4, a positive regulator of the TLR/NF-κB signaling pathway), and proinflammatory cytokines. In contrast, miR-21-5p inhibitor-transfected hDPCs upregulated the expression of TRAF6, PDCD4, and inflammatory cytokines following LPS stimulation. These findings suggest that miR-21-5p expression was induced by the NF-κB signaling pathway, which was in turn negatively regulated by miR-21-5p via downregulation of TRAF6 and PDCD4 expression in LPS-stimulated hDPCs.