Published papers

© 2018 American Society for Bone and Mineral Research Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell–cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell–cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

© 2018 The Authors The periosteum and mineralized bone are innervated by nerves that sense pain. These include both myelinated and unmyelinated neurons with either free nerve endings or bearing nociceptors. Parasympathetic and sympathetic autonomic nerves also innervate bone. However, little is known about the route sensory nerves take leaving the epiphyses of long bones at the adult knee joint. Here, we used transgenic mice that express fluorescent Venus protein in Schwann cells (Sox10-Venus mice) to visualize myelinated and unmyelinated nerves in the tibial epiphysis. Immunofluorescence to detect a pan-neuronal marker and the sensory neuron markers calcitonin gene-related peptide (CGRP) and tropomyosin receptor kinase A (TrkA) also revealed Schwann cell-associated sensory neurons. Foramina in the intercondylar area of the tibia were conserved between rodents and primates. Venus-labeled fibers were detected within bone marrow of the proximal epiphysis, exited through foramina along with blood vessels in the intercondylar area of the tibia, and joined Venus-labeled fibers of the synovial membrane and meniscus. These data suggest that innervation of the subchondral plate and trabecular bone within the tibial epiphysis carries pain signals from the knee joint to the brain through intercondylar foramina.

© 2019 American Society for Bone and Mineral Research Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+MSPC-derived adipogenesis in BM and increased LepR+MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

© 2019, The Author(s). Bone growth is linked to expansion of nearby organs, as is the case for the cranial base and the brain. Here, we focused on development of the mouse clivus, a sloping surface of the basioccipital bone, to define mechanisms underlying morphological changes in bone in response to brain enlargement. Histological analysis indicated that both endocranial and ectocranial cortical bone layers in the basioccipital carry the osteoclast surface dorsally and the osteoblast surface ventrally. Finite element analysis of mechanical stress on the clivus revealed that compressive and tensile stresses appeared mainly on respective dorsal and ventral surfaces of the basioccipital bone. Osteoclastic bone resorption occurred primarily in the compression area, whereas areas of bone formation largely coincided with the tension area. These data collectively suggest that compressive and tensile stresses govern respective localization of osteoclasts and osteoblasts. Developmental analysis of the basioccipital bone revealed the clivus to be angled in early postnatal wild-type mice, whereas its slope was less prominent in Tnfsf11 −/− mice, which lack osteoclasts. We propose that osteoclast-osteoblast “trans-pairing” across cortical bone is primarily induced by mechanical stress from growing organs and regulates shape and size of bones that encase the brain.

Objective: EphrinA2-EphA2 and ephrinB2-EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs). Material and Methods: The primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalisLPS at 0.001-10 μg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalisLPS at the optimal concentration (0.1 μg/mL) for different periods (6-48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse-transcription real-time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt-related transcription factor 2 (Runx2), and the osteoclastogenesis-related factors receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin were also evaluated. Results: The ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalisLPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased. Conclusion: The present study suggest that P. gingivalisLPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.

Background: We have previously revealed that omega-3 polyunsaturated fatty acids can prevent Helicobacter pylori infection by blocking the futalosine pathway, an alternative route for menaquinone (MK) biosynthesis. Materials and Methods: 1, Different H. pylori strains were grown in liquid media supplemented with linoleic acid, an omega-6 fatty acid, or its 10-hydroxy derivative, 10-hydroxy-cis-12-octadecenoic acid (HYA), in the presence or absence of MK. The bacterial numbers in the media were estimated by plating; 2, C57BL/6NCrl mice received drinking water supplemented with different fatty acids starting from 1 week before infection with H. pylori or Helicobacter suis until the end of the experiment. The gastric colonization levels of H. pylori or H. suis were determined 2 weeks after infection by plating or quantitative PCR, respectively; 3, Mice were given HYA, starting 1 week before infection with H. suis and continuing until 6 months after infection, for analysis of the gastric conditions. Results: 1, A low concentration (20 μmol/L) of HYA in culture broth suppressed the growth of H. pylori, and this inhibition was reduced by MK supplementation; 2, HYA treatment protected mice against H. pylori or H. suis infection; 3, HYA treatment suppressed the formation of lymphoid follicles in the gastric mucus layer after H. suis infection. Conclusions: HYA prevents gastric Helicobacter infections by blocking their futalosine pathways. Daily HYA supplementation is effective for the prevention of gastric mucosa-associated lymphoid tissue lymphoma induced by persistent infection with H. suis.

Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFPlow sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP- population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro. Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma.

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock-like syndrome. Human CD46-expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast-mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 105 colony-forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor-κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro-computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4+ and CD8+ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte-derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast-like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL's osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity.

Endochondral ossification is a developmental process by which cartilage is replaced by bone. Terminally differentiated hypertrophic chondrocytes are calcified, vascularized, and removed by chondroclasts before bone matrix is laid down by osteoblasts. In mammals, the malleus is one of three auditory ossicles that transmit vibrations of the tympanic membrane to the inner ear. The malleus is formed from a cartilaginous precursor without growth plate involvement, but little is known about how bones of this type undergo endochondral ossification. Here, we demonstrate that in the processus brevis of the malleus, clusters of osteoblasts surrounding the capillary loop produce bone matrix, causing the volume of the capillary lumen to decrease rapidly in post-weaning mice. Synchrotron X-ray tomographic microscopy revealed a concentric, cylindrical arrangement of osteocyte lacunae along capillaries, indicative of pericapillary bone formation. Moreover, we report that overexpression of Fosl1, which encodes a component of the AP-1 transcription factor complex, in osteoblasts significantly blocked malleal capillary narrowing. These data suggest that osteoblast/endothelial cell interactions control growth plate-free endochondral ossification through ‘osteogenic capillaries’ in a Fosl1-regulated manner.

The tyrosine kinase receptor, EphB4, mediates cross-talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin-B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over-expressing EphB4 under the control of collagen type-1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long-term culture-initiating cells (LTC-IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC-IC in vitro, where blocking EphB4/ephrin-B2 interactions decreased LTC-IC output. Similarly, short hairpin RNA-mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin-B2 expressing CD34+ HSC in LTC-IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin-1, IL-6, FLT-3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal-mediated support of hematopoiesis, and constitute a novel component of the HSC niche.

We have previously reported that transforming growth factor β (TGF-β) plays an essential role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde-assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE-seq and ChIP-seq) analyses indicated the cooperation of Smad2/3 with c-Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c-Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF-β, and c-Fos binding to open chromatin sites was suppressed by inhibition of TGF-β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c-Fos deficiency. These results suggest that TGF-β regulates RANKL-induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c-Fos.

January Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34+ hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34+ HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34+ HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Objective In vitro activation of the receptor EphB4 positively affects human osteoarthritis (OA) articular cell metabolism. However, the specific in vivo role of this ephrin receptor in OA remains unknown. We investigated in mice the in vivo effect of bone-specific EphB4 overexpression on OA pathophysiology. Methods Morphometric, morphologic, and radiologic evaluations were performed on postnatal day 5 (P5) mice and on 10-week-old mice. Knee OA was induced surgically by destabilization of the medial meniscus (DMM) in 10-week-old male EphB4 homozygous transgenic (EphB4-Tg) and wild-type (WT) mice. Medial compartment evaluations of cartilage were performed using histology and immunohistochemistry, and evaluations of subchondral bone using histomorphometry, osteoclast staining, and micro-computed tomography. Results There was no obvious phenotype difference in skeletal development between EphB4-Tg mice and WT mice at P5 or at 10 weeks. At 8 and 12 weeks post-DMM, the EphB4-Tg mice demonstrated significantly less cartilage alteration in the medial tibial plateau and the femoral condyle than did the WT mice. This was associated with a significant reduction of aggrecan and type II collagen degradation products, type X collagen, and collagen fibril disorganization in the operated EphB4-Tg mice. The medial tibial subchondral bone demonstrated a significant reduction in sclerosis, bone volume, trabecular thickness, and number of tartrate-resistant acid phosphatase-positive osteoclasts at both times assessed post-DMM in the EphB4-Tg mice than in the WT mice. Conclusion This is the first in vivo evidence that bone-specific EphB4 overexpression exerts a protective effect on OA joint structural changes. The findings of this study stress the in vivo importance of subchondral bone biology in cartilage integrity.

The tumor suppressor protein BRCA1 localizes to sites of DNA double-strand breaks (DSB), promoting repair by homologous recombination through the recruitment of DNA damage repair proteins. In normal cells, homologous recombination largely depends on BRCA1. However, assembly of the pivotal homologous recombination regulator RAD51 can occur independently of BRCA1 in the absence of 53BP1, another DNA damage response protein. How this assembly process proceeds is unclear, but important to understand in tumor cell settings where BRCA1 is disabled. Here we report that RNF8 regulates BRCA1-independent homologous recombination in 53BP1-depleted cells. RNF8 depletion suppressed the recruitment of RAD51 to DSB sites without affecting assembly or phosphorylation of the replication protein RPA in neocarzinostatin-treated or X-ray-irradiated BRCA1/53BP1-depleted cells. Furthermore, RNF8/BRCA1/53BP1-depleted cells exhibited less efficient homologous recombination than BRCA1/53BP1-depleted cells. Intriguingly, neither RNF8 nor its relative RNF168 were required for RAD51 assembly at DSB sites in 53BP1-expressing cells. Moreover, RNF8-independent RAD51 assembly was found to be regulated by BRCA1. Together, our findings indicate a tripartite regulation of homologous recombination by RNF8, BRCA1, and 53BP1. In addition, our results predict that RNF8 inhibition may be a useful treatment of BRCA1-mutated/53BP1low cancers, which are considered resistant to treatment by PARP1 inhibitors and of marked current clinical interest.