Overexpression of αCGRP promotes osteogenesis of periodontal ligament cells by regulation of YAP signaling

Human periodontal ligament cells (hPDLCs) are considered as an ideal cell type for periodontal tissue engineering as hPDLCs own mesenchymal stem cell-like properties. Additionally, it is suggested that α-calcitonin gene-related peptide (αCGRP) plays a pivotal role in the pathogenesis of periodontitis. However, the specific role of αCGRP on the regulation of alveolar bone regeneration which is essential for treatment of periodontitis remains unclear. In this study, lentiviral αCGRP expression vector was first transfected into hPDLCs. αCGRP expression and the osteogenesis-related gene (ALP, RUNX2, OCN, and BSP) expressions were detected. The results showed that expressions of osteogenic phenotypes were upregulated in αCGRP-transfected hPDLCs combined with an increased expression of Yes-associated protein (YAP), which is the key downstream effectors of Hippo pathway. Our observations suggest that αCGRP-mediated hPDLCs’ osteogenesis might relate with the activity of YAP signaling. These observations may reflect intrinsic functions of αCGRP in hPDLCs’ osteogenesis and its promising role in the treatment of bone deficiency in periodontal regeneration.     

HnRNPL inhibits the osteogenic differentiation of PDLCs stimulated by SrCl2 through repressing Setd2

Osteoporosis has been shown to intensify bone loss caused by periodontitis and both share common risk factors. One strategy utilized to manage the disease has been via the release of Sr ions by Strontium Ranelate having a direct effect on preventing osteoclast activation and promoting osteoblast differentiation. Previously we have developed and characterized porous Sr‐mesoporous bioactive glass (Sr‐MBG) scaffolds and demonstrated their ability to promote periodontal regeneration when compared to MBG alone. Our group further discovered a splicing factor, heterogeneous nuclear ribonucleoprotein L (hnRNPL), was drastically down‐regulated in periodontal ligament stem cells (PDLCs) stimulated by Sr through the activation of AKT pathway. Furthermore, hnRNPL restrained the osteogenic differentiation of PDLCs through down‐regulating H3K36me3‐specific methyltransferase Setd2. The goal of the present study was to investigate the mechanism of periodontal regeneration stimulated by Sr It was first found that the epigenetic mechanism of splicing factor hnRNPL participated in the osteogenesis processing of PDLCs stimulated by SrCl₂. Meanwhile, the different role of hnRNPL and SET domain containing 2 (Setd2) may provide some implication of the treatment of periodontitis patients simultaneously suffering from osteoporosis.     

Low-intensity pulsed ultrasound promotes bone morphogenic protein 9-induced osteogenesis and suppresses inhibitory effects of inflammatory cytokines on cellular responses via Rho-associated kinase 1 in human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low-intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9-induced osteogenic differentiation of PDLFs. In contrast, BMP2-induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS-induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS-PG), we also simultaneously treated PDLFs with LIPUS and LPS-PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK-binding kinase 1, and interferon regulatory factor 3 in LPS-PG-stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa-B ligand, and also diminished IL-1ß and tumor necrosis factor a (TNFa)-induced inflammatory reactions. Phosphorylation of Rho-associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1-specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS-PG-stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS-PG and IL-1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS-PG, IL-1ß, and TNFa and also promotes BMP9-induced osteogenesis through ROCK1 in PDLFs.     

Up-regulation of TβRIII facilitates the osteogenesis of supraspinous ligament-derived fibroblasts from patients with ankylosing spondylitis

Spinal supraspinous ligament (SL) osteogenesis is the key risk of ankylosing spondylitis (AS), with an unclear pathogenesis. We previously found that transforming growth factor β1 (TGF-β1), bone morphogenetic proteins (eg BMP2) and type III TGF-β1 receptor (TβRIII) expression were markedly up-regulated in AS-SLs. However, the roles of these closely related molecules in AS are unknown. Here, we showed that BMP2, TGF-β1, TβRIII and S100A4 (a fibroblast marker) were abundant in active osteogenic AS-SL tissues. In vitro, AS-SL fibroblasts (AS-SLFs) showed high BMP2, TGF-β1 and TβRIII expression and auto-osteogenic capacity. We further evaluated the role of TβRIII in the osteogenesis of normal SLFs. BMP2 combined with TGF-β1 induced the osteogenesis of TβRIII-overexpressing SLFs, but the activity was lost in SLFs upon TβRIII knockdown. Moreover, our data suggested that BMP2 combined with TGF-β1 significantly activated both TGF-β1/Smad signalling and BMP2/Smad/RUNX2 signalling to induce osteogenesis of SLFs with TβRIII up-regulation. Furthermore, our multi-strategy molecular interaction analysis approach indicated that TGF-β1 presented BMP2 to TβRIII, sequentially facilitating BMP2 recognition by BMPR1A and promoting the osteogenesis of TβRIII-overexpressing SLFs. Collectively, our results indicate that TGF-β1 combined with BMP2 may participate in the osteogenic differentiation of AS-SLF by acting on up-regulated TβRIII, resulting in excessive activation of both TGF-β1/Smad and BMP2/BMPR1A/Smad/RUNX2 signalling.     

Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2'-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.     

Epigenetic therapy attenuates oxidative stress in BMSCs during ageing

Oxidative stress, a hallmark of ageing, inhibits the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells in long bone. The dysfunction of the cellular antioxidant defence system is a critical cause of oxidative stress, but the mechanism of the decline of antioxidant defence in senescent stem cells remains elusive. Here, we found that EZH2, an epigenetic regulator of histone methylation, acted as a suppressor of the antioxidative defence system in BMSCs from the femur. The increased EZH2 led to a decrease in the levels of antioxidant enzymes and exaggerated oxidative damage in aged BMSCs, resulting in the defect of bone formation and regeneration. Mechanistically, EZH2 enhanced the modification of H3K27me3 on the promoter of Foxo1 and suppressed its function to activate the downstream genes in antioxidant defence. Moreover, epigenetic therapy targeting EZH2‐mediated H3K27me3 modification largely recovered the antioxidant defence in BMSCs and attenuate oxidative damage, leading to the recovery of the osteogenesis in old BMSCs. Taken together, our findings revealed novel crosstalk between histone epigenetic modification and oxidative stress during stem cell ageing, suggesting a possibility of epigenetic therapy in the recovery of BMSCs senescence and treatment of age‐related bone disease.     

MicroRNA expression profile of human periodontal ligament cells under the influence of Porphyromonas gingivalis LPS

Periodontitis is a chronic inflammatory disease which is caused by bacterial infection and leads to the destruction of periodontal tissues and resorption of alveolar bone. Thus, special attention should be paid to the mechanism under lipopolysaccharide (LPS)‐induced periodontitis because LPS is the major cause of periodontitis. However, to date, miRNA expression in the LPS‐induced periodontitis has not been well characterized. In this study, we investigated miRNA expression patterns in LPS‐treated periodontal ligament cells (PDLCs). Through miRNA array and differential analysis, 22 up‐regulated miRNAs and 28 down‐regulated miRNAs in LPS‐treated PDLCs were identified. Seven randomly selected up‐regulated (miR‐21‐5p, 498, 548a‐5p) and down‐regulated (miR‐495‐3p, 539‐5p, 34c‐3p and 7a‐2‐3p) miRNAs were examined by qRT‐PCR, and the results proved the accuracy of the miRNA array. Moreover, targets of these deregulated miRNAs were analysed using the miRWalk database. Database for Annotation, Visualization and Integration Discovery software were performed to analyse the Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway of differential expression miRNAs, and the results shown that Toll‐like receptor signalling pathway, cAMP signalling pathway, transforming growth factor‐beta signalling pathway, mitogen‐activated protein kinase (MAPK) signalling pathway and other pathways were involved in the molecular mechanisms underlying LPS‐induced periodontitis. In conclusion, this study provides clues for enhancing our understanding of the mechanisms and roles of miRNAs as key regulators of LPS‐induced periodontitis.     

MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network

MicroRNAs are a group of endogenous regulators that participate in several cellular physiological processes. However, the role of miR-137 in the osteogenic differentiation of human adipose-derived stem cells (hASCs) has not been reported. This study verified a general downward trend in miR-137 expression during the osteogenic differentiation of hASCs. MiR-137 knockdown promoted the osteogenesis of hASCs in vitro and in vivo. Mechanistically, inhibition of miR-137 activated the bone morphogenetic protein 2 (BMP2)-mothers against the decapentaplegic homolog 4 (SMAD4) pathway, whereas repressed lysine-specific histone demethylase 1 (LSD1), which was confirmed as a negative regulator of osteogenesis in our previous studies. Furthermore, LSD1 knockdown enhanced the expression of BMP2 and SMAD4, suggesting the coordination of LSD1 in the osteogenic regulation of miR-137. This study indicated that miR-137 negatively regulated the osteogenic differentiation of hASCs via the LSD1/BMP2/SMAD4 signaling network, revealing a new potential therapeutic target of hASC-based bone tissue engineering.     

Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway

Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25 ng/ml BMP2), BMP100 (induced with 100 ng/ml BMP2) and BMP25  + XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%–157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.     

RUNX2对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响

目的:研究和确认RUNX2在骨形态发生蛋白9(BMP9)诱导的间充质干细胞C3H10T1/2成骨分化中的作用。方法:通过Western blot、RT-PCR、荧光素酶活性分析检测BMP9对RUNX2表达的影响;分别在过表达RUNX2和RNA干扰抑制RUNX2表达的情况下,利用碱性磷酸酶(ALP)活性测定和染色、钙盐沉积实验,免疫细胞化学和裸鼠皮下异位成骨实验分析RUNX2对于BMP9诱导的间充质干细胞成骨分化的影响。结果:BMP9可以促进RUNX2的表达;RUNX2体外可促进BMP9诱导的C3H10T1/2的ALP活性和钙盐沉积,却抑制了OCN表达,RUNX2还可促进BMP9诱导的裸鼠皮下异位成骨;而在降低RUNX2表达后,BMP9诱导的C3H10T1/2细胞的ALP活性、钙盐沉积、OCN表达和裸鼠皮下异位成骨均受到抑制。结论:RUNX2可以促进BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化。     

Antagonistic and synergistic effects of bone morphogenetic protein 2/7 and all‐trans retinoic acid on the osteogenic differentiation of rat bone marrow stromal cells

The osteogenesis of bone marrow stromal cells (BMSCs) is of paramount importance for the repair of large‐size bone defects, which may be compromised by the dietary‐accumulated all‐trans retinoic acid (ATRA). We have shown that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) could induce bone regeneration in a significantly higher dose‐efficiency in comparison with homodimeric BMPs. In this study, we evaluated the effects of ATRA and BMP2/7 on the proliferation, differentiation, mineralization and osteogenic genes. ATRA and BMP2/7 exhibited both antagonistic and synergistic effects on the osteogenesis of BMSCs. ATRA significantly inhibited proliferation and expression of osteocalcin but enhanced the activity of alkaline phosphatase of BMSCs. On day 21, 50 ng/mL BMP2/7 could antagonize the inhibitive effects of ATRA and significantly enhance osteogenesis of BMSCs. These findings suggested a promising application potential of heterodimeric BMP2/7 in clinic to promote bone regeneration for the cases with dietary accumulated ATRA.     

Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology

Periodontal disease (Periodontitis) is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85%) and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro-inflammatory cytokine expression. This study demonstrated that Atp6v1c1 RNAi knockdown gene therapy mediated by AAV-shRNA-Atp6v1c1 is a promising novel therapeutic approach for the treatment of bone erosion and inflammatory related diseases, such as periodontitis and rheumatoid arthritis.     

Analysis of histone deacetylase inhibitor-induced responses in human periodontal ligament fibroblasts

Periodontal ligament (PDL) fibroblasts play critical roles in the regeneration of periodontal tissues damaged by periodontitis. Histone deacetylase inhibitors (HDIs) have been suggested to be potential tools in tissue engineering. The feasibility of using the HDI, sodium butyrate (NaB) for periodontal regeneration was examined by evaluating its effect on the osteogenic differentiation of human PDL fibroblasts and its modulation of the inflammatory responses to lipopolysaccharide (LPS). NaB did not cause significant cell death at 100 μM but promoted the expression of the osteoblast phenotype (Runx2, osterix, osteocalcin, and bone sialoprotein). NaB significantly inhibited the LPS-induced production of reactive oxygen species and the expression of pro-inflammatory cytokines (IL-1β and TNF-α). These results suggest that HDIs can offer a potential therapeutic agent for periodontal regeneration.     

XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

BMP2 (bone morphogenetic protein 2) is known to activate unfolded protein response signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation has not yet been elucidated. In this study, we demonstrate that BMP2 mediates mild endoplasmic reticulum stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of the XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5′-flanking regulatory region of the XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II collagen, type X collagen, and RUNX2; however, knockdown of XBP1S via the RNAi approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with RUNX2 and enhances RUNX2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of IHH (Indian hedgehog) and PTHrP (parathyroid hormone-related peptide). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by 1) acting as a cofactor of RUNX2 and 2) affecting IHH/PTHrP signaling.