IGF2 promotes alveolar bone regeneration in murine periodontitis via inhibiting cGAS/STING-mediated M1 macrophage polarization.

Periodontitis is a chronic inflammatory disease with the destruction of supporting periodontal tissue. This study evaluated the role of insulin-like growth factor 2 (IGF2) in periodontitis by inhibiting the polarization of M1 macrophages via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. IGF2 was enriched in the gingival tissue of murine periodontitis model identified by RNA sequencing. IGF2 application alleviated the expression of pro-inflammatory factors and promoted osteogenesis and the expression of related genes and proteins in a dose-dependent manner in periodontitis. The result of micro-CT verified this finding. Both in vivo and in vitro results revealed that IGF2 decreased the polarization of M1 macrophages and pro-inflammatory factors by immunofluorescence staining, flow cytometry, western blotting and RT-PCR. IGF2 application promoted the osteogenic ability of periodontal ligament fibroblasts (PDLFs) indirectly via its inhibition of M1 polarization evaluated by alkaline phosphatase and alizarin red staining. Then, the cGAS/STING pathway was upregulated in periodontitis and macrophages challenged by LPS, the inhibition of which led to downregulation of M1 polarization. Furthermore, IGF2 could downregulate cGAS, STING and the phosphorylation of P65. Collectively, our study indicates IGF2 can regulate the polarization of M1 macrophages via the cGAS/STING pathway and highlights the promising future of IGF2 as a therapeutic treatment for periodontitis.

HIF-1α regulates osteoclastogenesis and alveolar bone resorption in periodontitis via ANGPTL4.

OBJECTIVE: The mechanism of alveolar bone resorption caused by periodontitis is not fully understood. We sought to investigate whether microenvironmental changes of local hypoxia are involved in these processes. METHODS: In this study, periodontitis models of control mice and knockout of Hypoxia Induced Factor 1α (HIF-1α) harboring Cathepsin K (CTSK) Cre mice were constructed to study the effect of osteoclasts affected by hypoxic environment on alveolar bone resorption. RAW264.7 cells were subsequently induced by CoCl2 to observe the effects of HIF-1α and Angiopoietin-like Protein 4 (ANGPTL4) on osteoblast differentiation and fusion. RESULTS: The degree of alveolar bone resorption in the periodontitis tissues was lesser in mice with conditional knockout of HIF-1α in osteoclasts than in wild-type mice. We also observed that HIF-1α conditional knockout mice had fewer osteoclasts on the alveolar bone surface than control mice. HIF-1α increases the expression of ANGPTL4 and promotes the differentiation of RAW264.7 cells into osteoblasts and cell fusion under chemically simulated hypoxic conditions. CONCLUSION: HIF-1α regulates osteoclastogenesis and participates in bone resorption in periodontitis through ANGPTL4.

Hypoxia-induced factor 1α promotes osteotomy-induced regional acceleratory phenomenon via DC-STAMP mediated membrane fusion.

OBJECTIVES: The mechanisms of regional acceleratory phenomenon (RAP) induced by orthognathic osteotomy are unclear. It was not known if locally hypoxic microenvironment changes were involved in this phenomenon. METHODS: Hypoxia-induced factor-1α knockout mice harboring Cathepsin K (CTSK) Cre were used to investigate the effect of hypoxia-driven osteoclasts on alveolar bone remodeling. RAW264.7 cells were induced by CoCl2 to observe the effects of dendritic cell-specific transmembrane protein (DC-STAMP) on the fusion and differentiation of osteoclasts. RESULTS: We found mandibular osteotomy of C57 mice induced active alveolar osteoclasts and increased hypoxia-induced factor-1α (HIF-1α) positive staining areas. Alveolar bone density of the 10-week-old HIF-1α conditional knockout (CKO) mouse was increased at 10 and 14 days after bilateral mandibular osteotomy. Moreover, decreased numbers of osteoclasts and matrix metalloproteinase 9 (MMP-9)-positive cells were observed on the surface of bone resorption lacunae in the CKO group. HIF-1α could increase the expression level of DC-STAMP to enhance osteoclastogenesis and cell fusion in active RAW264.7 cells. CONCLUSION: Our data considered hypoxia-driven osteoclasts resorption to be an adaptive mechanism to permit alveolar bone loss after bilateral mandibular osteotomy of mice.

HIF-1α mediates osteoclast-induced disuse osteoporosis via cytoophidia in the femur of mice.

Osteoporosis induced by disuse because of bed rest or the aerospace industry has become one of the most common skeletal disorders. However, mechanisms underlying the disuse osteoporosis remain largely unknown. We validated the tail-suspended model in mice and demonstrated that there is bone loss in the trabecular and cortical bones of the femur. Importantly, we showed that genetical deletion of hypoxia-inducible factor-1α (HIF-1α) in osteoclasts ameliorated osteoclastic bone resorption in the trabecular bone whereas pharmacological treatment with HIF-1α inhibitor protected the hindlimb-unloaded mice from disuse-induced osteoporosis in the trabecular and cortical bones. The HIF-1α knockout RAW264.7 cells and RNA-sequencing proved that HIF-1α is vital for osteoclastogenesis and bone resorption because it regulated the level of inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase (CTPS) via cellular myelocytomatosis (c-Myc) oncogene. The IMPDH and CTPS are vital nucleotide metabolic enzymes which have an important functional role in cell metabolism, and they can assemble into intracellular linear or ring-shaped structures to cope with cell stress. Interestingly, both in vitro and in vivo, the IMPDH and CTPS cytoophidia were found in osteoclasts, and the level of HIF-1α correlated with osteoclastogenesis and bone-resorbing activity. Our data revealed that HIF-1α/c-Myc/cytoophidia signalling might be required for osteoclasts to mediate cell metabolism in disuse-induced osteoporosis. Overall, our results revealed a new role of HIF-1α/c-Myc/cytoophidia in supporting osteoclastogenesis and bone resorption and exposed evidence for its role in the pathogenesis of disuse osteoporosis, which might provide promising therapeutic targets.

LncRNA-mRNA Expression Profiles of Osteoclast After Conditional Knockout HIF-1α.

Background: Osteoclasts, which are multinucleated cells formed by monocyte fusion, play a key role in bone resorption. Hypoxia-inducible factor (HIF)-1α is vital for the development of osteoclasts in hypoxic environments and during bone resorption. However, additional research is required to further study the HIF-1α-dependent regulation of osteoclast differentiation at the genetic level. Methods: In our study, RNA sequencing (RNA-seq) was used to identify the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in conditional HIF-1α-knockout osteoclasts. Results: A total of 1,320 mRNAs and 95 lncRNAs were differentially expressed. The expression of lncRNAs MSTRG.7566.12 and MSTRG.31769.2 were strongly negatively correlated with that of Mmp9, Ctsk, etc. Conclusion: Our research provides a basis for further understanding the role of mRNAs and lncRNAs in conditional HIF-1α-knockout osteoclasts, and many of these molecules may be potential targets for treating bone diseases related to HIF-1α.