Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and ...osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.
Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating ...osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast-osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.
Abstract
Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells that play different roles in different organs by sensing surrounding environmental factors. Initially, it was thought that ...ILC2s in bone marrow (BM) are progenitors for systemic ILC2s, which migrate to other organs and acquire effector functions. However, accumulating evidence that ILC2s differentiate in peripheral tissues suggests that BM ILC2s may play a specific role in the BM as a unique effector per se. Here, we demonstrate that BM ILC2s highly express the receptor activator of nuclear factor κB ligand (RANKL), a robust cytokine for osteoclast differentiation and activation, and RANKL expression on ILC2s is up-regulated by interleukin (IL)-2, IL-7 and all-trans retinoic acid (ATRA). BM ILC2s co-cultured with BM-derived monocyte/macrophage lineage cells (BMMs) in the presence of IL-7 induce the differentiation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in a RANKL-dependent manner. In contrast, BM ILC2s stimulated with IL-33 down-regulate RANKL expression and convert BMMs differentiation into M2 macrophage-like cells rather than osteoclasts by granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-13 production. Intravital imaging using two-photon microscopy revealed that a depletion of ILC2s prominently impaired in vivo osteoclast activity in an IL-7 plus ATRA-induced bone loss mouse model. These results suggest that ILC2s regulate osteoclast activation and contribute to bone homeostasis in both steady state and IL-33-induced inflammation.
How ILC2s regulate osteoclastogenesis
Intravital microscopy with multiphoton excitation is a recently developed optical imaging technique for deep tissue imaging without fixation or sectioning, which permits examination of fundamental ...concepts regarding the dynamic nature of cells under physiological and pathological conditions in living animals. This novel technique also offers exciting opportunities for pharmacological research by providing new platforms for the study of cellular dynamics in response to drugs in vivo. Moreover, fluorescent chemical probes for functional or molecular analysis in single cells in vivo play important roles in pharmacology. For example, we have recently revealed the pharmacodynamic actions of different biological agents for the treatment of rheumatoid arthritis (RA) in vivo by directly visualizing drug-induced cellular behaviors and functions of osteoclasts on bone surfaces. This review focuses on the principles and advantages of intravital imaging for the dissection of pharmacological mechanisms, and discusses how such imaging can contribute to the drug development process, introducing recent trials that evaluated the in vivo pharmacological effects of various agents.
Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial-temporal relationship and mode of ...interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell-cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell-cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell-cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.
Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing loss and brain abscess formation. However, the ...pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a single-cell RNA sequencing analysis on human cholesteatoma samples and identify a pathogenic fibroblast subset characterized by abundant expression of inhibin βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in these fibroblasts results in decreased osteoclast differentiation in a murine model of cholesteatoma. Moreover, follistatin, an antagonist of activin A, reduces osteoclastogenesis and resultant bone erosion in cholesteatoma. Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone destruction via the induction of local osteoclastogenesis, suggesting a potential therapeutic target.
Anti-resorptive drugs are widely used for the treatment of osteoporosis, but excessive inhibition of osteoclastogenesis can suppress bone turnover and cause the deterioration of bone quality. Sialic ...acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a transmembrane protein expressed on osteoclast precursor cells and mature osteoclasts. Siglec-15 regulates proteins containing immunoreceptor tyrosine-based activation motif (ITAM) domains, which then induce nuclear factor of activated T-cells 1 (NFATc1), a master transcription factor of osteoclast differentiation. Anti-Siglec-15 antibody modulates ITAM signaling in osteoclast precursors and inhibits the maturation of osteoclasts in vitro. However, in situ pharmacological effects, particularly during postmenopausal osteoporosis, remain unclear. Here, we demonstrated that anti-Siglec-15 antibody treatment protected against ovariectomy-induced bone loss by specifically inhibiting the generation of multinucleated osteoclasts in vivo. Moreover, treatment with anti-Siglec-15 antibody maintained bone formation to a greater extent than with risedronate, the first-line treatment for osteoporosis. Intravital imaging revealed that anti-Siglec-15 antibody treatment did not cause a reduction in osteoclast motility, whereas osteoclast motility declined following risedronate treatment. We evaluated osteoclast activity using a pH-sensing probe and found that the bone resorptive ability of osteoclasts was lower following anti-Siglec-15 antibody treatment compared to after risedronate treatment. Our findings suggest that anti-Siglec-15 treatment may have potential as an anti-resorptive therapy for osteoporosis, which substantially inhibits the activity of osteoclasts while maintaining physiological bone coupling.
•Bone loss following ovariectomy was prevented by anti-Siglec-15 antibody.•Anti-Siglec-15 antibody inhibited multinucleation of osteoclasts in vivo.•Treatment with anti-Siglec-15 antibody maintained bone formation.
AIM: To differentiate focal liver lesions based on enhancement patterns using three-dimensional ultrasonography (3D US) with perflubutane-based contrast agent.METHODS: Two hundred and eighty two ...patients with focal liver lesions,including 168 hepatocellular carcinomas (HCCs),63 metastases,40 hemangiomas and 11 focal nodular hyperplasias (FNHs),were examined by 3D US with perflubutane-based contrast agent.Tomographic ultrasound images and sonographic angiograms were reconstructed.Among 282 lesions,enhancemen...
Sonazoid(Daiichi Sankyo,Tokyo,Japan),a secondgeneration of a lipid-stabilized suspension of a perfluorobutane gas microbubble contrast agent,has been used clinically in patients with liver tumors and ...for harmonic gray-scale ultrasonography(US)in Japan since January 2007.Sonazoid-enhanced US has two phases of contrast enhancement:vascular and late.In the late phase of Sonazoid-enhanced US,we scanned the whole liver using this modality at a low mechanical index(MI)without destroying the microbubbles, and this method allows detection of small viable hepatocellular carcinoma(HCC)lesions which cannot be detected by conventional US as perfusion defects in the late phase.Re-injection of Sonazoid into an HCC lesion which previously showed a perfusion defect in the late phase is useful for confirming blood flow intothe defects.High MI intermittent imaging at 2 frames per second in the late phase is also helpful in differentiation between necrosis and viable hypervascular HCC lesions.Sonazoid-enhanced US by the coded harmonic angio mode at a high MI not only allows clear observation of tumor vessels and tumor enhancement, but also permits automatic scanning with Sonazoidenhanced three dimensional(3D)US.Fusion images combining US with contrast-enhanced CT or contrastenhanced MRI have made it easy to detect typical or atypical HCC lesions.By these methods,Sonazoidenhanced US can characterize liver tumors,grade HCC lesions histologically,recognize HCC dedifferentiation, evaluate the efficacy of ablation therapy or transcatheter arterial embolization,and guide ablation therapy for unresectable HCC.This article reviews the current developments and applications of Sonazoid-enhanced US and Sonazoid-enhanced 3D US for diagnosing and treating hepatic lesions,especially HCC.
PDF
