l-arginine promotes angio-osteogenesis to enhance oxidative stress-inhibited bone formation by ameliorating mitophagy.

Background: Osteoporosis is one of the most common bone diseases in middle-aged and elderly populations worldwide. The development of new drugs to treat the disease is a key focus of research. Current treatments for osteoporosis are mainly directed at promoting osteoblasts and inhibiting osteoclasts. However, there is currently no ideal approach for osteoporosis treatment. l-arginine is a semi-essential amino acid involved in a number of cellular processes, including nitric production, protein biosynthesis, and immune responses. We previously reported that l-arginine-derived compounds can play a regulatory role in bone homeostasis. Purpose: To investigate the specific effect of l-arginine on bone homeostasis. Methods: Mildly aged and ovariectomized mouse models were used to study the effects of l-arginine on osteogenesis and angiogenesis, assessed by micro-computed tomography and immunostaining of bone tissue. The effect of l-arginine on osteogenesis, angiogenesis, and adipogenesis was further studied in vitro using osteoblasts obtained from cranial cap bone, endothelial cells, and an adipogenic cell line. Specific methods to assess these processes included lipid staining, cell migration, tube-forming, and wound-healing assays. Protein and mRNA expression was determined for select biomarkers. Results: We found that l-arginine attenuated bone loss and promoted osteogenesis and angiogenesis. l-arginine increased the activity of vascular endothelial cells, whereas it inhibited adipogenesis in vitro. In addition, we found that l-arginine altered the expression of PINK1/Parkin and Bnip3 in the mitochondria of osteoblast-lineage and endothelial cells, thereby promoting mitophagy and protecting cells from ROS. Similarly, l-arginine treatment effectively ameliorated osteoporosis in an ovariectomized mouse model. Conclusion: l-arginine promotes angio-osteogenesis, and inhibits adipogenesis, effects mediated by the PINK1/Parkin- and Bnip3-mediated mitophagy. The Translational Potential of this Article: L-arginine supplementation may be an effective adjunct therapy in the treatment of osteoporosis.

Urolithin B reduces cartilage degeneration and alleviates osteoarthritis by inhibiting inflammation.

Osteoarthritis is the most prevalent degenerative joint disease reported worldwide. Conventional treatment strategies mainly focus on medication and involve surgical joint replacement. The use of these therapies is limited by gastrointestinal complications and the lifespan of joint prostheses. Hence, safe and efficacious drugs are urgently needed to impede the osteoarthritis progression. Urolithin B, a metabolite of ellagic acid in the gut, exhibits anti-inflammatory and antioxidant properties; however, its role in osteoarthritis remains unclear. In this study, we demonstrated that urolithin B efficiently inhibits the inflammatory factor-induced production of matrix metalloproteinases (MMP3 and MMP13) in vitro and upregulates the expression of type II collagen and aggrecan. Urolithin B alleviates cartilage erosion and osteophyte formation induced by anterior cruciate ligament transections. Moreover, urolithin B inhibits the activation of the NF-κB pathway by reducing the phosphorylation of Iκb-α and the nuclear translocation of P65. In summary, urolithin B significantly inhibits inflammation and alleviates osteoarthritis. Hence, urolithin B can be considered a potential agent suitable for the effective treatment of osteoarthritis in the future.

Restrained Mitf-associated autophagy by Mulberroside A ameliorates osteoclastogenesis and counteracts OVX-Induced osteoporosis in mice.

Bone and mineral metabolism homeostasis accounts for the maintenance of normal skeletal remodeling. However, with aging and changes in hormone levels, over-activated osteoclasts disrupt homeostasis, induce osteoporosis, and even cause osteoporotic fractures, leading to an enormous economic burden. Despite the rapid development of pharmacological therapy for osteoporosis, safer and more effective treatments remain to be explored. Here, we demonstrate that Mulberroside A (Mul-A), a natural component extracted from mulberry bark and branches, effectively suppresses osteoclastogenesis in vitro and counteracts bone loss caused by ovariectomy (OVX). The mechanism underlying this effect involves the repression of autophagic flux during osteoclastogenesis by Mul-A, which can be attributed to the restrained expression of microphthalmia-related transcription factor (Mitf) and its nuclear translocation. Importantly, Mitf overexpression partially reverses the inhibitory effects of Mul-A on autophagy and osteoclastogenesis. Moreover, applying two autophagy agonizts, rapamycin and Torin 1, attenuates the osteoclastogenic regulatory role of Mul-A. Collectively, our study demonstrates that Mul-A damages osteoclast differentiation and ameliorates osteoporosis caused by estrogen deficiency by modulation of Mitf-associated autophagy, indicating its therapeutic potential against osteoporosis.

USP7 Inhibits Osteoclastogenesis via Dual Effects of Attenuating TRAF6/TAK1 Axis and Stimulating STING Signaling.

Ubiquitination is a reversible post-translational modification implicated in cell differentiation, homeostasis, and organ development. Several deubiquitinases (DUBs) decrease protein ubiquitination through the hydrolysis of ubiquitin linkages. However, the role of DUBs in bone resorption and formation is still unclear. In this study, we identified DUB ubiquitin-specific protease 7 (USP7) as a negative regulator of osteoclast formation. USP7 combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits its ubiquitination by impairing the Lys63-linked polyubiquitin chain. Such impairment leads to the suppression of receptor activator of NF-κB ligand (RANKL)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation without affecting TRAF6 stability. USP7 also protects the stimulator of interferon genes (STING) against degradation, inducing interferon-ß (IFN-ß) expression in osteoclast formation, thereby inhibiting osteoclastogenesis cooperatively with the classical TRAF6 pathway. Furthermore, USP7 inhibition accelerates osteoclast differentiation and bone resorption both in vitro and in vivo. Contrarily, USP7 overexpression impairs osteoclast differentiation and bone resorption in vitro and in vivo. Additionally, in ovariectomy (OVX) mice, USP7 levels are lower than those in sham-operated mice, suggesting that USP7 plays a role in osteoporosis. Altogether, our data reveal the dual effect of USP7-mediated TRAF6 signal transduction and USP7-mediated protein degradation of STING in osteoclast formation.

ERK1 loss accelerates the progression of osteoarthritis in aged mice via NRF2/BACH1 signaling.

Osteoarthritis (OA) is now a common degenerative joint related disease. However, the clinical efficacy of drugs associated with cartilage regeneration remains limited. In our study, we firstly explored the role of ERK1 in the progression of OA. We clarified that ERK1-deficient mice were susceptible to age-related OA. The higher OARSI scores and more severe cartilage degeneration was observed in the ERK1-deficient mice. ERK1 deficiency decreased the nuclear transportation of Nrf2 in the chondrocytes and accelerated chondrocyte aging in vitro. Moreover, chondrocytes with ERK1 deficiency elevated the nuclear expression of BACH1, resulting in lowered expression of antioxidant enzymes in ERK1-deficient chondrocytes. The Nrf2 activator dimethyl fumarate (DMF) was used. Our experiments demonstrated the protective function of DMF against OA in ERK1 knockout mice. Above all, we confirmed the effects of ERK1 on the progression of OA and clarified the mechanisms underlying these effects. DMF might has significant use in the development of novel drugs for the therapy of OA in the future.

USP1 inhibition suppresses the progression of osteosarcoma via destabilizing TAZ.

Mutations and altered expression of deubiquitinating enzymes (DUBs) profoundly influence tumor progression. Ubiquitin-specific protease 1 (USP1) is a well-characterized human DUB reportedly overexpressed in and associated with maintaining the mesenchymal stem cell status of osteosarcoma (OS); however, the potential mechanisms of USP1 in OS remain poorly understood. In this study, we identified that USP1 directly interacts with Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) in OS cell lines, and with mechanistic analysis indicating that the anti-OS effects of USP1 inhibition could be partially attributed to TAZ instability, with its reduced nuclear accumulation responsible for a subsequent decrease in the expression of downstream genes associated with the Hippo signaling pathway. Moreover, pharmacological inhibition USP1 by ML323 presented the similar effects on Hippo signaling pathway and suppressed OS growth and metastasis both in vitro and in vivo. Taken together, our results revealed a novel molecular mechanism underlying the function of USP1 in OS and a potential role of ML323 as a therapeutic strategy for the clinical treatment of OS.

Urolithin A Promotes Angiogenesis and Tissue Regeneration in a Full-Thickness Cutaneous Wound Model.

The treatment of chronic wound is an important topic of current clinical issue. Neovascularization plays a crucial role in skin wound healing by delivering fresh nutrients and oxygen to the wound area. The aim of this study was to investigate the mechanisms of urolithin A (UA) in angiogenesis during wound healing. The results of in vitro experiments showed that treatment with UA (5-20 µM) promoted the proliferation, migration, and angiogenic capacity of HUVECs. Furthermore, we investigated the effect of UA in vivo using a full-thickness skin wound model. Subsequently, we found that UA promoted the regeneration of new blood vessels, which is consistent with the results of accelerated angiogenesis in vitro experiments. After UA treatment, the blood vessels in the wound are rapidly formed, and the deposition and remodeling process of the collagen matrix is also accelerated, which ultimately promotes the effective wound healing. Mechanistic studies have shown that UA promotes angiogenesis by inhibiting the PI3K/AKT pathway. Our study provides evidence that UA can promote angiogenesis and skin regeneration in chronic wounds, especially ischemic wounds.

Mechanical force promotes dimethylarginine dimethylaminohydrolase 1-mediated hydrolysis of the metabolite asymmetric dimethylarginine to enhance bone formation.

Mechanical force is critical for the development and remodeling of bone. Here we report that mechanical force regulates the production of the metabolite asymmetric dimethylarginine (ADMA) via regulating the hydrolytic enzyme dimethylarginine dimethylaminohydrolase 1 (Ddah1) expression in osteoblasts. The presence of -394 4 N del/ins polymorphism of Ddah1 and higher serum ADMA concentration are negatively associated with bone mineral density. Global or osteoblast-specific deletion of Ddah1 leads to increased ADMA level but reduced bone formation. Further molecular study unveils that mechanical stimulation enhances TAZ/SMAD4-induced Ddah1 transcription. Deletion of Ddah1 in osteoblast-lineage cells fails to respond to mechanical stimulus-associated bone formation. Taken together, the study reveals mechanical force is capable of down-regulating ADMA to enhance bone formation.

The SFRP1 Inhibitor WAY-316606 Attenuates Osteoclastogenesis Through Dual Modulation of Canonical Wnt Signaling.

Osteoporosis, a noteworthy age-related disease induced by imbalanced osteogenesis and osteoclastogenesis, is a serious economic burden on both individuals and society. Small molecule drugs with dual effects on both bone resorption and mineralization are pressingly needed. Secreted frizzled-related protein 1 (SFRP1), a well-known extracellular repressor of canonical Wnt signaling, has been reported to regulate osteogenesis. Global SFRP1 knockout mice show significantly elevated bone mass. Although osteoclasts (OCs) express and secrete SFRP1, the role of SFRP1 produced by OCs in osteoclastogenesis and osteoporosis remains unclear. In this work, the levels of SFRP1 were found to be increased in patients with osteoporosis compared with healthy controls. Pharmacological inhibition of SFRP1 by WAY-316606 (WAY)- attenuated osteoclastogenesis and bone resorption in vitro. The expressions of OC-specific genes were suppressed by the SFRP1 inhibitor, WAY. Mechanistically, both extracellular and intracellular SFRP1 could block activation of the canonical Wnt signaling pathway, and WAY reverse the silent status of canonical Wnt through dual effects, leading to osteoclastogenesis inhibition and osteogenesis promotion. Severe osteopenia was observed in the ovariectomized (OVX) mouse model, and WAY treatment effectively improved the OVX-induced osteoporosis. In summary, this work found that SFRP1 supports OC differentiation and function, which could be attenuated by WAY through dual modulation of canonical Wnt signaling, suggesting its therapeutic potential. © 2021 American Society for Bone and Mineral Research (ASBMR).

EIF4A3-induced circular RNA PRKAR1B promotes osteosarcoma progression by miR-361-3p-mediated induction of FZD4 expression.

Emerging evidence indicates that circRNAs are broadly expressed in osteosarcoma (OS) cells and play a crucial role in OS progression. Recently, cancer-specific circRNA circPRKAR1B has been identified by high-throughput sequencing and is recorded in publicly available databases. Nevertheless, the detailed functions and underlying mechanisms of circPRKAR1B in OS remains poorly understood. By functional experiments, we found that circPRKAR1B enhanced OS cell proliferation, migration, and promotes OS epithelial-mesenchymal transition (EMT). Mechanistic investigations suggested that circPRKAR1B promotes OS progression through sponging miR-361-3p to modulate the expression of FZD4. Subsequently, we identified that EIF4A3 promoted cirPRKAR1B formation through binding to the downstream target of circPRKAR1B on PRKAR1B mRNA. Further rescue study revealed that overexpression of the Wnt signalling could impair the onco-suppressor activities of the silencing of circPRKAR1B. Interestingly, further experiments indicated that circPRKAR1B is involved in the sensitivity of chemoresistance in OS. On the whole, our results demonstrated that circPRKAR1B exerted oncogenic roles in OS and suggested the circPRKAR1B/miR-361-3p/FZD4 axis plays an important role in OS progression and might be a potential therapeutic target.

DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling.

Osteoporosis-related fractures, such as femoral neck and vertebral fractures, are common in aged people, resulting in increased disability rate and health-care costs. Thus, it is of great importance to clarify the mechanism of osteoclast-related osteoporosis and find effective ways to avoid its complication. In this study, gene expression profile analysis and real-time polymerase chain reaction revealed that DUSP6 expression was suppressed in human and mice osteoporosis cases. In vitro experiments confirmed that DUSP6 overexpression prevented osteoclastogenesis, whereas inhibition of DUSP6 by small interference RNA or with a chemical inhibitor, (E/Z)-BCI, had the opposite effect. (E/Z)-BCl significantly accelerated the bone loss process in vivo by enhancing osteoclastogenesis. Bioinformatics analyses and in vitro experiments indicated that miR-181a was an upstream regulator of DUSP6. Moreover, miR-181a positively induced the differentiation and negatively regulated the apoptosis of osteoclasts via DUSP6. Furthermore, downstream signals by ERK2 and SMAD2 were also found to be involved in this process. Evaluation of ERK2-deficiency bone marrow-derived macrophages confirmed the role of ERK2 signaling in the DUSP6-mediated osteoclastogenesis. Additionally, immunoprecipitation assays confirmed that DUSP6 directly modified the phosphorylation status of SMAD2 and the subsequent nuclear transportation of NFATC1 to regulate osteoclast differentiation. Altogether, this study demonstrated for the first time the role of miRNA-181a/DUSP6 in the progression of osteoporosis via the ERK2 and SMAD2 signaling pathway. Hence, DUSP6 may represent a novel target for the treatment of osteoclast-related diseases in the future.

An Antioxidant Sesquiterpene Inhibits Osteoclastogenesis Via Blocking IPMK/TRAF6 and Counteracts OVX-Induced Osteoporosis in Mice.

Excessive bone resorption induced by increased osteoclast activity in postmenopausal women often causes osteoporosis. Although the pharmacological treatment of osteoporosis has been extensively developed, a safer and more effective treatment is still needed. Here, we found that curcumenol (CUL), an antioxidant sesquiterpene isolated from Curcuma zedoaria, impaired receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro, whereas the osteoblastogenesis of MC3T3-E1 cells was not affected. We further demonstrated that CUL treatment during RANKL-induced osteoclastogenesis promotes proteasomal degradation of TRAF6 by increasing its K48-linked polyubiquitination, leading to suppression of mitogen-activated protein kinases (MAPKs) and NF-κB pathways and the production of reactive oxygen species (ROS). We also showed that inositol polyphosphate multikinase (IPMK) binds with TRAF6 to reduce its K48-linked polyubiquitination under RANKL stimulation. Concurrently, IPMK deficiency inhibits osteoclast differentiation. The binding between IPMK and TRAF6 blocked by CUL treatment was found in our study. Finally, we confirmed that CUL treatment prevented ovariectomy (OVX)-induced bone loss in mice. In summary, our study demonstrates that CUL could impair the stability of TRAF6 enhanced by IPMK and suppress excessive osteoclast activity in estrogen-deficient mice to treat osteoporosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

miR-21-5p targets SKP2 to reduce osteoclastogenesis in a mouse model of osteoporosis.

Osteoporosis results from an imbalance between bone formation and bone resorption. Traditional drugs for treating osteoporosis are associated with serious side effects, and thus, new treatment methods are required. This study investigated the role of differentially expressed microRNAs during osteoclast differentiation and osteoclast activity during osteoarthritis as well as the associated underlying mechanisms. We used a microarray to screen microRNAs that decreased in the process of osteoclast differentiation and verified miR-21-5p to decrease significantly using RT-qPCR. In follow-up experiments, we found that miR-21-5p targets SKP2 to regulate osteoclast differentiation. In vivo, ovariectomized mice were used to simulate perimenopausal osteoporosis induced by estrogen deficiency, and miR-21-5p treatment inhibited bone resorption and maintained bone cortex and trabecular structure. These results suggest that miR-21-5p is a new therapeutic target for osteoporosis.

ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/ß-catenin signalling pathways.

Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel-like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small-molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose-dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound-healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial-mesenchymal transition phenotype, including N-cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10-day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.