Metabolic reprogramming in skeletal cell differentiation.

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions. From the beginning steps of chondrogenesis that prefigures most of the skeleton, to the rapid bone accrual during skeletal growth, followed by bone remodeling of the mature skeleton, cell differentiation is integral to skeletal health. While growth factors and nuclear proteins that influence skeletal cell differentiation have been extensively studied, the role of cellular metabolism is just beginning to be uncovered. Besides energy production, metabolic pathways have been shown to exert epigenetic regulation via key metabolites to influence cell fate in both cancerous and normal tissues. In this review, we will assess the role of growth factors and transcription factors in reprogramming cellular metabolism to meet the energetic and biosynthetic needs of chondrocytes, osteoblasts, or osteoclasts. We will also summarize the emerging evidence linking metabolic changes to epigenetic modifications during skeletal cell differentiation.

Investigating Causal Effects of Hematologic Traits on Lung Cancer: A Mendelian Randomization Study.

BACKGROUND: Observational studies have suggested blood cell counts may act as predictors of cancer. It is not known whether these hematologic traits are causally associated with lung cancer. METHODS: Two-sample bidirectional univariable Mendelian randomization (MR) and multivariable MR (MVMR) were performed to investigate the causal association between hematologic traits and the overall risk of lung cancer and three histologic subtypes [lung adenocarcinoma, squamous cell lung cancer, and small cell lung cancer (SCLC)]. The instrumental variables of 23 hematologic traits were strictly selected from large-scale genome-wide association studies. Inverse-variance weighted method and five extra methods were used to obtain robust causal estimates. RESULTS: We found evidence that genetically influenced higher hematocrit [OR, 0.845; 95% confidence interval (CI), 0.783-0.913; P = 1.68 × 10-5] and hemoglobin concentration (OR, 0.868; 95% CI, 0.804-0.938; P = 3.20 × 10-4) and reticulocyte count (OR, 0.923; 95% CI, 0.872-0.976; P = 5.19 × 10-3) decreased lung carcinoma risk, especially in ever smokers. MVMR further identified hematocrit independently of smoking as an independent predictor. Subgroup analysis showed that a higher plateletcrit level increased the risk of small cell lung carcinoma (OR, 1.288; 95% CI, 1.126-1.474; P = 2.25 × 10-4). CONCLUSIONS: Genetically driven higher levels of reticulocyte count and hematocrit decreased lung cancer risk. Higher plateletcrit had an adverse effect on SCLC. Hematologic traits may act as low-cost factors for lung cancer risk stratification. IMPACT: Further studies are required to elucidate the potential mechanisms underlying the dysregulation of homeostasis related to hematologic traits, such as subclinical inflammation.

Pharmacological inhibition of protein S-palmitoylation suppresses osteoclastogenesis and ameliorates ovariectomy-induced bone loss.

Background: Excessive osteoclast formation disrupts bone homeostasis, thereby significantly contributing to pathological bone loss associated with a variety of diseases. Protein S-palmitoylation is a reversible post-translational lipid modification catalyzed by ZDHHC family of palmitoyl acyltransferases, which plays an important role in various physiological and pathological processes. However, the role of palmitoylation in osteoclastogenesis has never been explored. Consequently, it is unclear whether this process can be targeted to treat osteolytic bone diseases that are mainly caused by excessive osteoclast formation. Materials and methods: In this study, we employed acyl-biotin exchange (ABE) assay to reveal protein S-palmitoylation in differentiating osteoclasts (OCs). We utilized 2-bromopalmitic acid (2-BP), a pharmacological inhibitor of protein S-palmitoylation, to inhibit protein palmitoylation in mouse bone marrow-derived macrophages (BMMs), and tested its effect on receptor activator of nuclear factor κß ligand (RANKL)-induced osteoclast differentiation and activity by TRAP staining, phalloidin staining, qPCR analyses, and pit formation assays. We also evaluated the protective effect of 2-BP against estrogen deficiency-induced bone loss and bone resorption in ovariectomized (OVX) mice using µCT, H&E staining, TRAP staining, and ELISA assay. Furthermore, we performed western blot analyses to explore the molecular mechanism underlying the inhibitory effect of 2-BP on osteoclastogenesis. Results: We found that many proteins were palmitoylated in differentiating OCs and that pharmacological inhibition of palmitoylation impeded RANKL-induced osteoclastogenesis, osteoclast-specific gene expression, F-actin ring formation and osteoclastic bone resorption in vitro, and to a lesser extent, osteoblast formation from MC3T3-E1 cells. Furthermore, we demonstrated that administration of 2-BP protected mice from ovariectomy-induced osteoporosis and bone resorption in vivo. Mechanistically, we showed that 2-BP treatment inhibited osteoclastogenesis partly by downregulating the expression of c-Fos and NFATc1 without overtly affecting RANKL-induced activation of osteoclastogenic AKT, MAPK, and NF-κB pathways. Conclusion: Pharmacological inhibition of palmitoylation potently suppresses RANKL-mediated osteoclast differentiation in vitro and protects mice against OVX-induced osteoporosis in vivo. Mechanistically, palmitoylation regulates osteoclast differentiation partly by promoting the expression of c-Fos and NFATc1. Thus, palmitoylation plays a key role in promoting osteoclast differentiation and activity, and could serve as a potential therapeutic target for the treatment of osteoporosis and other osteoclast-related diseases. The translational potential of this article: The translation potential of this article is that we first revealed palmitoylation as a key mechanism regulating osteoclast differentiation, and therefore provided a potential therapeutic target for treating osteolytic bone diseases.

The emerging studies on mesenchymal progenitors in the long bone.

Mesenchymal progenitors (MPs) are considered to play vital roles in bone development, growth, bone turnover, and repair. In recent years, benefiting from advanced approaches such as single-cell sequence, lineage tracing, flow cytometry, and transplantation, multiple MPs are identified and characterized in several locations of bone, including perichondrium, growth plate, periosteum, endosteum, trabecular bone, and stromal compartment. However, although great discoveries about skeletal stem cells (SSCs) and progenitors are present, it is still largely obscure how the varied landscape of MPs from different residing sites diversely contribute to the further differentiation of osteoblasts, osteocytes, chondrocytes, and other stromal cells in their respective destiny sites during development and regeneration. Here we discuss recent findings on MPs' origin, differentiation, and maintenance during long bone development and homeostasis, providing clues and models of how the MPs contribute to bone development and repair.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

Teriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

A bibliometric analysis of Wnt signaling pathway: from the top-100 cited articles to emerging trends.

BACKGROUND: Wnt signaling pathway plays a vital role in the regulation of development. An increasing number of articles about Wnt pathway components have been published. By analyzing these studies' characteristics and qualities, we aim to reveal the current research focus and emerging trends in Wnt signaling. METHODS: The databases of Web of Science Core Collection, BIOSIS Citation Index, MEDLINE, etc. were utilized to identify articles on May 23rd, 2020. Wnt signaling pathway-related articles were identified, the 100 most cited articles and articles in the last decade were selected and calculated for citations without self-citation. The subsequent analysis included citation density (citations/article age), time-related flux, authorship, institution, journal, geographic distribution, and theme. RESULTS: These articles were published mainly from 2000 to 2009 (62%). Citations per article ranged from 599 to 3,780 with a median number of 880 times. Most studies (66%) came from the United States. Nusse Roel and Clevers Hans (15 and 13 papers) have contributed significantly to the field. The most highlighted study themes were cancer (15%), embryo development (14%), and cytoplasm signal transduction (11%). From 2011 to 2020, interest in emerging subtopics, including osteogenesis, immune, apoptosis, autophagy, microRNA, and cancer stem cell, are rising. CONCLUSIONS: Cancer, embryo development, stem cell, and signal transduction process still play a major role in the field. With multiple emerging subtopics and investigation on an integrated view of the Wnt signal network, the association of Wnt with diseases was further revealed.

Old unreduced obturator dislocation of the hip: A case report.

BACKGROUND: Obturator dislocation is a rare type of hip dislocation, accounting for about 2%-5% of all hip dislocations. The occurrence of old unreduced obturator dislocation is even more infrequent, with only 17 cases reported in nine studies, most of which were from the 1950s to 1980s in developing countries. CASE SUMMARY: A 38-year-old woman from Hunan Province, China presented with stiffness of the left hip in abduction, flexion, and external rotation after falling from a 2-meter-tall tree onto her left knee 1.5 mo prior. Pelvic radiograph and computed tomography revealed obturator dislocation of the left hip accompanied by impaction fracture at the superolateral aspect of the left femoral head without associated acetabulum fracture. Open reduction was performed, resulting in restoration of the concentric alignment of the left hip. After surgery, 6-wk skin traction was applied and the patient was kept in bed for an additional 2 wk. At 3 mo after surgery, the patient reported experiencing some pain, which did not affect the function of the affected limb, and some movement restriction but no abduction deformity or claudication was present. An X-ray showed that the left hip was homocentric, and there was no sign of posttraumatic arthritis or avascular necrosis. CONCLUSION: Open reduction may be an effective treatment strategy for the rare condition of old unreduced obturator dislocation with short neglect time.

Strontium-doped gelatin scaffolds promote M2 macrophage switch and angiogenesis through modulating the polarization of neutrophils.

The immune system mediates inflammation, vascularization and the first response to injuries or implanted biomaterials. Although the function of neutrophils in tissue repair has been extensively studied, its complete role in the tissue regeneration of biomaterials, specifically the resolution of inflammation and promotion of angiogenesis, is unclear. Here, we fabricate nanofibrous gelatin scaffolds containing 10% (w/w) strontium-hydroxyapatite (SrHA) via phase-separation methods to investigate Sr-mediated regulation of neutrophil polarization and, subsequently, the effects on angiogenesis and macrophage polarization. Compared with neutrophils cultured on pure gelatin or HA-incorporated gelatin scaffolds, neutrophils on SrHA-incorporated gelatin scaffolds show more N2 polarization in vitro and in vivo and significantly greater production of immunomodulatory and angiogenic factors. The Sr-induced immunomodulatory and proangiogenic functions of neutrophils are mediated through NF-κB pathway downregulation and increased STAT3 phosphorylation. Thus, neutrophils play a vital role in tissue engineering, and Sr-incorporated scaffolds efficiently promote neutrophil polarization to the N2 phenotype, enhancing resolution of inflammation and ultimately promoting angiogenesis and tissue regeneration. Thus, incorporation of neutrophils in analyses of the immune characteristics of scaffolds and the development of immunomodulatory biomaterials that can regulate neutrophils are novel and promising strategies in tissue engineering.

The regulatory effect of has-circ-0001146/miR-26a-5p/MNAT1 network on the proliferation and invasion of osteosarcoma.

Osteosarcoma is a malignant bone tumour with the lowest survival rates out of all paediatric cancers and is primarily diagnosed in children and adolescents. MNAT1 is a subunit in the cyclin-dependent kinase-activating kinase complex. Abnormal up-regulation of MNAT1 has been associated with the poor prognosis of multiple cancers. Bioinformatics analysis showed that has-circ-0001146 and miR-26a-5p were involved in the regulation of MNAT1 in osteosarcoma. The present study investigated the regulatory effects of has-circ-0001146 and miR-26a-5p on MNAT1 expression using luciferase reporter and RNA-pull down assays. The effects of the has-circ-0001146/miR26a-5p/Mnat1 network on the proliferation and invasion of osteosarcoma were evaluated by cell viability, apoptosis, migration, and invasion assays. Osteosarcoma tissues showed higher MNAT1 and has-circ-0001146 expression than adjacent normal tissues, although the expression of MNAT1 was not significantly up-regulated in sarcomas according to TCGA databases. As indicated by luciferase reporter and RNA-pull down assays, miR-26a-5p was able to bind to both has-circ-0001146 and MNAT1 mRNA. The depletion of has-circ-0001146 as well as the increase of miR-26a-5p decreased MNAT1 expression in osteosarcoma cells, while the reduction of miR-26a-5p was associated with increased MNAT1 expression. These data suggested that has-circ-0001146 promoted MNAT1 expression by competitively binding to miR-26a-5p with MNAT1 mRNA. The depletion of has-circ-0001146 or MNAT1 or the increase of miR-26a-5p inhibited osteosarcoma cell viability and invasion, and increased apoptosis. Reduction of miR-26a-5p conversely promoted osteosarcoma cell viability and invasion. The present study confirmed that has-circ-0001146 blocked miR-26a-5p targeting MNAT1 in osteosarcoma cells, thereby promoting the malignant behaviours of osteosarcoma cells.

The top 100 most cited articles on total hip arthroplasty: a bibliometric analysis.

BACKGROUND: Over the past few decades, more and more articles about total hip arthroplasty have been published. We noticed, however, little is known about the characteristics and qualities of these studies. METHODS: The databases of Web of Science Core Collection, BIOSIS Citation Index, MEDLINE, etc. were utilized for the identification of articles published from 1990 to May 2019. Total hip arthroplasty-related articles were identified, and the 100 most cited articles were selected for subsequent analysis of citation count, citation density (citations/article age), authorship, theme, geographic distribution, time-related flux, level of evidence, and network analysis. RESULTS: The selected 100 articles were published mainly in the 1990s (46%) and 2000s (47%) with almost equal amount. Citations per article ranged from 994 to 191. Leading countries were the USA followed by Canada, England, and Sweden, all located in North America and Western Europe. The most highlighted study themes were postoperative thrombosis and surgical methods and materials. The most common level of evidence was level III (35%). The network analysis connoted that radiography, acetabulum, reoperation, and bone cement had a high degree of centrality in the 1990s, while cement had a high degree of centrality in the 2000s and 2010s. CONCLUSIONS: The time, area, and theme distribution of the top 100 most cited articles in the total hip arthroplasty have been thoroughly analyzed. It is noticeable that postoperative thromboembolism currently plays a major role in the field of total hip arthroplasty researches. However, most of them focus on the effectiveness of different treatments and drugs; little is known about its underlying mechanisms and influencing factors.

MAT1 facilitates the lung metastasis of osteosarcoma through upregulation of AKT1 expression.

AIMS: We aimed to elucidate the effects and mechanisms of MAT1 in the progression of osteosarcoma, especially for its lung metastasis. MAIN METHODS: CCK-8 and flow cytometry assays were carried out to detect the proliferation and apoptosis of osteosarcoma cells. Wound healing and transwell assays were used to determine cell migration and invasion abilities. Real time quantitative PCR (RT-PCR) and western blot technologies were applied to detect the expression levels of RNA and protein, respectively. KEY FINDS: The results showed that both the mRNA and protein expression levels of MAT1 were elevated in osteosarcoma tissues with lung metastasis and metastatic lung tissues, particularly in the metastatic lung tissues, as compared to the osteosarcoma tissues without lung metastasis. High expression level of MAT1 in osteosarcoma patients showed a negative association with the overall survival. In addition, upregulation of MAT1 induced significant increases in cell growth, migration and invasion and an obvious inhibition in cell apoptosis in osteosarcoma MG63 and 143B cells, as well as elevated AKT1 expression level. Moreover, knockdown of AKT1 obviously impaired MAT1-mediated promotions in cell migration and invasion in vitro, as well as repressed tumor growth and reduced the number of metastatic lung tumors in xenografted nude mice. SIGNIFICANCE: This study reveals that high expression of MAT1 closely related to the poor prognosis and malignant clinical process of osteosarcoma patients. MAT1 serves as a promoter in the lung metastasis of osteosarcoma through increasing AKT1 expression. Our study may provide a potent therapeutic target for the lung metastasis of osteosarcoma.

Gli1 identifies osteogenic progenitors for bone formation and fracture repair.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1+ cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1+ cells residing immediately beneath the growth plate, termed here "metaphyseal mesenchymal progenitors" (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of ß-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1+ cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair.

Deferoxamine synergizes with transforming growth factor-β signaling in chondrogenesis

Abstract Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, is an epidemic disease that affects millions of people worldwide. Despite extensive recent work on the cellular biology of osteoarthritis, the precise mechanisms involved are still poorly understood and there is no effective treatment for this disease. The role of transforming growth factor-beta (TGF-β) in promoting chondrogenesis and inducing the expression of cartilage-specific extracellular matrix molecules to form cartilage is well-established. Historically, TGF-β has been considered to prevent osteoarthritis, but recent work suggests that TGF-β overexpression accelerates the progression of osteoarthritis in vivo. Clinically, it is therefore important to limit TGF-β expression while still providing effective treatment of osteoarthritis. One possible approach to achieve this effect would be to use a combination of TGF-β with other small molecular chemical compounds. Hypoxia promotes chondrogenesis and the usefulness of deferoxamine, a chelating agent that mimics hypoxia, in stimulating chondrogenesis has been investigated in clinical trials. In this study, we investigated the role of deferoxamine in TGF-β-induced chondrogenesis in pre-chondrogenic cells and examined whether deferoxamine synergizes with the TGF-β signaling pathway to promote chondrocyte differentiation.

Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice.

Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmp1-Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmp1 gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmp1-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmp1-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmp1-Cre in the gastrointestinal mesenchyme, deletion of Bmpr1a with Dmp1-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmp1-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types.

LRG1 modulates invasion and migration of glioma cell lines through TGF-ß signaling pathway.

Studies have shown that the abnormal expression of leucine-rich α2 glycoprotein 1 (LRG1) is associated with multiple malignancies, yet its role in glioma pathology remains to be elucidated. In this study, we investigated the role of LRG1 in regulating proliferation, migration and invasion of glioma cells by establishing glioma cell strains with constitutively silenced or elevated LRG1 expression. LRG1 overexpression and silenced cell lines demonstrated modulation of glioma cellular proliferation, migration and invasion through MTT, cell scratching and Transwell assays. Furthermore, overexpression of LRG1 led to augmented activation of transforming growth factor-ß (TGF-ß) signaling pathway as well as downregulation of E-cadherin and resultant enhanced invasiveness, which was reversed by TGF-ß signaling pathway inhibitor SB431542. In summary, our findings suggest that LRG1 promotes invasion and migration of glioma cells through TGF-ß signaling pathway.

Stable knockdown of LRG1 by RNA interference inhibits growth and promotes apoptosis of glioblastoma cells in vitro and in vivo.

Leucine-rich α2 glycoprotein 1 (LRG1) has been shown to be aberrantly expressed in multiple human malignancies. However, the biological functions of LRG1 in human glioblastoma remain unknown. Here, we report for the first time the role of LRG1 in glioblastoma development based on the preliminary in vitro and in vivo data. We first confirmed the expression of LRG1 in human glioblastoma cell lines. Next, to investigate the role of LRG1 in the tumorigenesis and development of glioblastoma, a short hairpin RNA (shRNA) construct targeting LRG1 mRNA was transfected into U251 glioblastoma cells to generate a cell line with stably silenced LRG1 expression. The results showed that silencing of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at G0/G1 phase, and enhanced apoptosis in U251 cells in vitro. Consistently, LRG1 silencing resulted in the downregulation of key cell cycle factors including cyclin D1, B, and E and apoptotic gene Bcl-2 while elevated the levels of pro-apoptotic Bax and cleaved caspase-3, as determined by Western blot analysis. We further demonstrate that the silencing of LRG1 expression effectively reduced the tumorigenicity of U251 cells, delayed tumor formation, and promoted apoptosis in a xenograft tumor model in vivo. In conclusion, silencing the expression of LRG1 suppresses the growth of glioblastoma U251 cells in vitro and in vivo, suggesting that LRG1 may play a critical role in glioblastoma development, and it may have potential clinical implications in glioblastoma therapy.