Publications by authors named "Yousef Abu-Amer"

65 Publications

Actions of the NLRP3 and NLRC4 inflammasomes overlap in bone resorption.

FASEB J 2021 09;35(9):e21837

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO, USA.

Overwhelming evidence indicates that excessive stimulation of innate immune receptors of the NOD-like receptor (NLR) family causes significant damage to multiple tissues, yet the role of these proteins in bone metabolism is not well known. Here, we studied the interaction between the NLRP3 and NLRC4 inflammasomes in bone homeostasis and disease. We found that loss of NLRP3 or NLRC4 inflammasome attenuated osteoclast differentiation in vitro. At the tissue level, lack of NLRP3, or NLRC4 to a lesser extent, resulted in higher baseline bone mass compared to wild-type (WT) mice, and conferred protection against LPS-induced inflammatory osteolysis. Bone mass accrual in mutant mice correlated with lower serum IL-1β levels in vivo. Unexpectedly, the phenotype of Nlrp3-deficient mice was reversed upon loss of NLRC4 as bone mass was comparable between WT mice and Nlrp3;Nlrc4 knockout mice. Thus, although bone homeostasis is perturbed to various degrees by the lack of NLRP3 or NLRC4, this tissue appears to function normally upon compound loss of the inflammasomes assembled by these receptors.
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http://dx.doi.org/10.1096/fj.202100767RRDOI Listing
September 2021

Methods to Analyze the Roles of TAK1, TRAF6, and NEMO in the Regulation of NF-κB Signaling by RANK Stimulation During Osteoclastogenesis.

Methods Mol Biol 2021 ;2366:267-282

Department of Orthopedic Surgery, Washington University School of Medicine and Shriners Hospital for Children, Saint Louis, MO, USA.

The skeletal system is constantly undergoing turnover in order to create strong, organized structures, requiring the bone breakdown and building properties by osteoclasts and osteoblasts, respectively. However, in pathological disease states, excessive osteoclast activity can cause bone loss leading to increase in morbidity and mortality. Osteoclasts differentiate from macrophages in the presence of various factors. M-CSF is a cytokine that is required to maintain the survival of macrophages. However, RANKL is the critical factor required for differentiation of osteoclasts. RANKL is produced from a variety of different cell types such as osteoblasts and osteocytes. RANKL binds to RANK, its receptor, on the surface of osteoclast precursors, which activates various signaling pathways to drive the transcription and production of genes important for osteoclast formation. The major signaling pathway activated by RANKL-RANK interaction is the NF-κB pathway. The NF-κB pathway is the principle inflammatory response pathway activated by a variety of stimuli such as inflammatory cytokines, genotoxic stress, and other factors. This likely explains the finding that inflammatory diseases often present with some component of increased osteoclast formation and activity, driving bone loss. Determining the signaling mechanisms downstream of RANKL can provide valuable therapeutic targets for the treatment of bone loss in various disease states.
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http://dx.doi.org/10.1007/978-1-0716-1669-7_16DOI Listing
January 2021

Targeting angiogenesis for fracture nonunion treatment in inflammatory disease.

Bone Res 2021 Jun 7;9(1):29. Epub 2021 Jun 7.

Department of Orthopaedic Surgery, School of Medicine, Washington University, St. Louis, MO, USA.

Atrophic fracture nonunion poses a significant clinical problem with limited therapeutic interventions. In this study, we developed a unique nonunion model with high clinical relevance using serum transfer-induced rheumatoid arthritis (RA). Arthritic mice displayed fracture nonunion with the absence of fracture callus, diminished angiogenesis and fibrotic scar tissue formation leading to the failure of biomechanical properties, representing the major manifestations of atrophic nonunion in the clinic. Mechanistically, we demonstrated that the angiogenesis defect observed in RA mice was due to the downregulation of SPP1 and CXCL12 in chondrocytes, as evidenced by the restoration of angiogenesis upon SPP1 and CXCL12 treatment in vitro. In this regard, we developed a biodegradable scaffold loaded with SPP1 and CXCL12, which displayed a beneficial effect on angiogenesis and fracture repair in mice despite the presence of inflammation. Hence, these findings strongly suggest that the sustained release of SPP1 and CXCL12 represents an effective therapeutic approach to treat impaired angiogenesis and fracture nonunion under inflammatory conditions.
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http://dx.doi.org/10.1038/s41413-021-00150-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184936PMC
June 2021

Intra-articular silencing of periostin via nanoparticle-based siRNA ameliorates post-traumatic osteoarthritis in mice.

Arthritis Rheumatol 2021 May 13. Epub 2021 May 13.

Department of Orthopaedic Surgery, Musculoskeletal Research Center, Washington University, School of Medicine, St. Louis, MO, United States.

Objective: Recent evidence delineates an emerging role of Periostin (Postn) in osteoarthritis (OA) as its expression subsequent to knee injury is detrimental to the articular cartilage. We hypothesize that intra-articular knockdown of Postn in a murine model of post-traumatic OA would ameliorate OA.

Methods: Post-traumatic OA was induced in 10-week-old male C57BL/6J mice (n=24) by destabilization of the medial meniscus (DMM) and analyzed 8-week post-surgery. Intra-articular Postn was inhibited by siRNA using a novel peptide-nucleotide polyplex. Cartilage degeneration (OARSI score) and synovitis were assessed histologically. Bone changes were measured by μCT. The effect and mechanism of Postn silencing were investigated in human chondrocytes treated with IL-1β with or without IKK2 inhibitor, SC-514.

Results: Peptide-siRNA nanoplatform significantly abolished Postn expression. OARSI score was significantly less in mice receiving Postn siRNA (10.94±0.66) compared to both untreated (22.38±1.30,P=0.002) and scrambled siRNA (22.69±0.87,P=0.002) treatment. No differences were observed in synovitis. Subchondral bone sclerosis, BV/TV, vBMD, and heterotopic ossification were significantly low in Postn siRNA treatment. Immunostaining of cartilage revealed that Postn knockdown reduced the DMM-induced MMP-13 intensity, phosphorylation of p65, and immunoreactivity of aggrecan neoepitope, DIPEN. Postn knockdown also suppressed IL-1β-induced MMP-13 and ADAMTS-4 in chondrocytes. Mechanistically, Postn-induced MMP-13 was abrogated by SC-514 demonstrating a link between Postn and NF-κB.

Conclusion: Intra-articular delivery of Postn siRNA nanocomplex represents a promising clinical approach to mitigate the severity of joint degeneration and provides an unequivocal scientific rationale for longitudinal studies. Employing a cartilage-specific gene knockout strategy will further illuminate the functional role of Postn in OA.
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http://dx.doi.org/10.1002/art.41794DOI Listing
May 2021

Enhanced Tendon-to-Bone Healing via IKKβ Inhibition in a Rat Rotator Cuff Model.

Am J Sports Med 2021 03 28;49(3):780-789. Epub 2021 Jan 28.

Department of Biomedical Engineering, Columbia University, New York, New York, USA.

Background: More than 450,000 rotator cuff repairs are performed annually, yet healing of tendon to bone often fails. This failure is rooted in the fibrovascular healing response, which does not regenerate the native attachment site. Better healing outcomes may be achieved by targeting inflammation during the early period after repair. Rather than broad inhibition of inflammation, which may impair healing, the current study utilized a molecularly targeted approach to suppress IKKβ, shutting down only the inflammatory arm of the nuclear factor κB (NF-κB) signaling pathway.

Purpose: To evaluate the therapeutic potential of IKKβ inhibition in a clinically relevant model of rat rotator cuff repair.

Study Design: Controlled laboratory study.

Methods: After validating the efficacy of the IKKβ inhibitor in vitro, it was administered orally once a day for 7 days after surgery in a rat rotator cuff repair model. The effect of treatment on reducing inflammation and improving repair quality was evaluated after 3 days and 2, 4, and 8 weeks of healing, using gene expression, biomechanics, bone morphometry, and histology.

Results: Inhibition of IKKβ attenuated cytokine and chemokine production in vitro, demonstrating the potential for this inhibitor to reduce inflammation in vivo. Oral treatment with IKKβ inhibitor reduced NF-κB target gene expression by up to 80% compared with a nontreated group at day 3, with a subset of these genes suppressed through 14 days. Furthermore, the IKKβ inhibitor led to enhanced tenogenesis and extracellular matrix production, as demonstrated by gene expression and histological analyses. At 4 weeks, inhibitor treatment led to increased toughness, no effects on failure load and strength, and decreases in stiffness and modulus when compared with vehicle control. At 8 weeks, IKKβ inhibitor treatment led to increased toughness, failure load, and strength compared with control animals. IKKβ inhibitor treatment prevented the bone loss near the tendon attachment that occurred in repairs in control.

Conclusion: Pharmacological inhibition of IKKβ successfully suppressed excessive inflammation and enhanced tendon-to-bone healing after rotator cuff repair in a rat model.

Clinical Relevance: The NF-κB pathway is a promising target for enhancing outcomes after rotator cuff repair.
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http://dx.doi.org/10.1177/0363546520985203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8464217PMC
March 2021

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head.

J Bone Miner Res 2021 02 2;36(2):357-368. Epub 2020 Nov 2.

Center for Excellence in Hip Disorders, Scottish Rite for Children, Dallas, TX, USA.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4191DOI Listing
February 2021

Radiation causes tissue damage by dysregulating inflammasome-gasdermin D signaling in both host and transplanted cells.

PLoS Biol 2020 08 6;18(8):e3000807. Epub 2020 Aug 6.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri, United Sates of America.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.
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http://dx.doi.org/10.1371/journal.pbio.3000807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446913PMC
August 2020

LDHA-mediated ROS generation in chondrocytes is a potential therapeutic target for osteoarthritis.

Nat Commun 2020 07 9;11(1):3427. Epub 2020 Jul 9.

Department of Orthopaedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.

The contribution of inflammation to the chronic joint disease osteoarthritis (OA) is unclear, and this lack of clarity is detrimental to efforts to identify therapeutic targets. Here we show that chondrocytes under inflammatory conditions undergo a metabolic shift that is regulated by NF-κB activation, leading to reprogramming of cell metabolism towards glycolysis and lactate dehydrogenase A (LDHA). Inflammation and metabolism can reciprocally modulate each other to regulate cartilage degradation. LDHA binds to NADH and promotes reactive oxygen species (ROS) to induce catabolic changes through stabilization of IκB-ζ, a critical pro-inflammatory mediator in chondrocytes. IκB-ζ is regulated bi-modally at the stages of transcription and protein degradation. Overall, this work highlights the function of NF-κB activity in the OA joint as well as a ROS promoting function for LDHA and identifies LDHA as a potential therapeutic target for OA treatment.
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http://dx.doi.org/10.1038/s41467-020-17242-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347613PMC
July 2020

Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO.

Elife 2020 03 23;9. Epub 2020 Mar 23.

Department of Orthopaedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, United States.

Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-κB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKγ/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions.
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http://dx.doi.org/10.7554/eLife.56095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145425PMC
March 2020

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway.

JCI Insight 2020 02 13;5(3). Epub 2020 Feb 13.

Department of Orthopaedic Surgery, School of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA.

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair.
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http://dx.doi.org/10.1172/jci.insight.131816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098799PMC
February 2020

Mechanisms Underlying Bone Loss Associated with Gut Inflammation.

Int J Mol Sci 2019 Dec 15;20(24). Epub 2019 Dec 15.

Department of Orthopaedic Surgery and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MI 63110, USA.

Patients with gastrointestinal diseases frequently suffer from skeletal abnormality, characterized by reduced bone mineral density, increased fracture risk, and/or joint inflammation. This pathological process is characterized by altered immune cell activity and elevated inflammatory cytokines in the bone marrow microenvironment due to disrupted gut immune response. Gastrointestinal disease is recognized as an immune malfunction driven by multiple factors, including cytokines and signaling molecules. However, the mechanism by which intestinal inflammation magnified by gut-residing actors stimulates bone loss remains to be elucidated. In this article, we discuss the main risk factors potentially contributing to intestinal disease-associated bone loss, and summarize current animal models, illustrating gut-bone axis to bridge the gap between intestinal inflammation and skeletal disease.
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http://dx.doi.org/10.3390/ijms20246323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940820PMC
December 2019

PARP1 Hinders Histone H2B Occupancy at the NFATc1 Promoter to Restrain Osteoclast Differentiation.

J Bone Miner Res 2020 04 7;35(4):776-788. Epub 2020 Jan 7.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO, USA.

Induction of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) is essential for macrophage differentiation into osteoclasts (OCs), but the underlying mechanisms remain unclear. The ability of poly(ADP-ribose) polymerase 1 (PARP1) to poly-ADP-ribosylate NFATc1 in T cells prompted us to investigate the PARP1 and NFATc1 interaction during osteoclastogenesis. However, extensive studies failed to directly link PARP1 to NFATc1. A combination of transcriptomics and proteomics studies was then used to identify PARP1 targets under these conditions. These unbiased approaches in conjunction with site-directed mutagenesis studies revealed that PARP1 inhibited NFATc1 expression and OC formation by ADP-ribosylating histone H2B at serine 7 and decreasing the occupancy of this histone variant at the NFATc1 promoter. The anti-osteoclastogenic function of PARP1 was confirmed in vivo in several mouse models of PARP1 loss-of-function or gain-of-function, including a novel model in which PARP1 was conditionally ablated in myeloid cells. Thus, PARP1 ADP-ribosylates H2B to negatively regulate NFATc1 expression and OC differentiation. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465553PMC
April 2020

Inflammatory Responses Reprogram T Through Impairment of Neuropilin-1.

Sci Rep 2019 07 18;9(1):10429. Epub 2019 Jul 18.

Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Chronic inflammatory insults compromise immune cell responses and ultimately contribute to pathologic outcomes. Clinically, it has been suggested that bone debris and implant particles, such as polymethylmethacrylate (PMMA), which are persistently released following implant surgery evoke heightened immune, inflammatory, and osteolytic responses that contribute to implant failure. However, the precise mechanism underlying this pathologic response remains vague. T, the chief immune-suppressive cells, express the transcription factor Foxp3 and are potent inhibitors of osteoclasts. Using an intra-tibial injection model, we show that PMMA particles abrogate the osteoclast suppressive function of T. Mechanistically, PMMA particles induce T instability evident by reduced expression of Foxp3. Importantly, intra-tibial injection of PMMA initiates an acute innate immune and inflammatory response, yet the negative impact on T by PMMA remains persistent. We further show that PMMA enhance T17 response at the expense of other T effector cells (T), particularly T1. At the molecular level, gene expression analysis showed that PMMA particles negatively regulate Nrp-1/Foxo3a axis to induce T instability, to dampen T activity and to promote phenotypic switch of T to T17 cells. Taken together, inflammatory cues and danger signals, such as bone and implant particles exacerbate inflammatory osteolysis in part through reprogramming T.
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http://dx.doi.org/10.1038/s41598-019-46934-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639378PMC
July 2019

Stem cell-derived extracellular vesicles attenuate the early inflammatory response after tendon injury and repair.

J Orthop Res 2020 01 26;38(1):117-127. Epub 2019 Jul 26.

Department of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8233, St. Louis, Missouri.

Adipose-derived stem cells (ASCs) have the potential to enhance tendon repair via paracrine regulation of the inflammatory response to injury. Extracellular vesicles (EVs), which are secreted by ASCs, have shown promise in mediating this process. This study was designed to evaluate the effect of ASC EVs on early tendon healing using a mouse Achilles tendon injury and repair model. EVs were isolated from the conditioned medium of naïve and interferonγ-primed ASCs and applied to the repair site via a collagen sheet. Tendon healing was assessed in nuclear factor-κB (NF-κB)-luciferase reporter mice up to 7 days after suture repair. As anticipated, repair site NF-κB activity increased greater than twofold following tendon repair. Treatment with EVs from primed but not naïve ASCs effectively suppressed the response. Accordingly, the pro-inflammatory genes Il1b and Ifng were both dramatically increased in repaired tendons, while primed, but not naïve ASC EVs attenuated the response. Compared with control repairs, primed ASC EVs further reduced the rate of post-repair tendon gap formation and rupture and facilitated collagen formation at the injury site. Additional experiments demonstrated that EVs target macrophages and that primed ASC EVs were most effective in blocking macrophage NF-κB activity. Collectively, the findings of this study demonstrate that primed ASC EVs, similar to ASCs, attenuate the early tendon inflammatory response after injury via modulation of the macrophage inflammatory response. Statement of clinical significance: These findings introduce a new cell-free therapy, derived from stem cells, for tendon repair with the potential for improved therapeutic efficacy and safety. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:117-127, 2020.
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http://dx.doi.org/10.1002/jor.24406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917960PMC
January 2020

Attenuation of NF-κB in Intestinal Epithelial Cells Is Sufficient to Mitigate the Bone Loss Comorbidity of Experimental Mouse Colitis.

J Bone Miner Res 2019 10 25;34(10):1880-1893. Epub 2019 Jul 25.

Department of Orthopaedic Surgery and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA.

Skeletal abnormalities are common comorbidities of inflammatory bowel disease (IBD). Patients suffering from IBD, including ulcerative colitis and Crohn's disease, present with skeletal complications. However, the mechanism underpinning IBD-associated bone loss remains vague. Intestinal inflammation generates an inflammatory milieu at the intestinal epithelium that leads to dysregulation of mucosal immunity through gut-residing innate lymphoid cells (ILCs) and other cell types. ILCs are recently identified mucosal cells considered as the gatekeeper of gut immunity and their function is regulated by intestinal epithelial cell (IEC)-secreted cytokines in response to the inflammatory microenvironment. We first demonstrate that serum as well as IECs collected from the intestine of dextran sulfate sodium (DSS)-induced colitis mice contain high levels of inflammatory and osteoclastogenic cytokines. Mechanistically, heightened inflammatory response of IECs was associated with significant intrinsic activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in IECs and increased frequency of ILC1, ILC3, and myeloid osteoclast progenitors. Validating the central role of IEC-specific NF-κB activation in this phenomenon, conditional expression of constitutively active inhibitor kappa B kinase 2 (IKK2) in IECs in mice recapitulates the majority of the cellular, inflammatory, and osteolytic phenotypes observed in the chemically induced colitis. Furthermore, conditional deletion of IKK2 from IECs significantly attenuated inflammation and bone loss in DSS-induced colitis. Finally, using the DSS-induced colitis model, pharmacologic inhibition of IKK2 was effective in reducing frequency of ILC1 and ILC3 cells, attenuated circulating levels of inflammatory cytokines, and halted colitis-associated bone loss. Our findings identify IKK2 in IECs as viable therapeutic target for colitis-associated osteopenia.
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http://dx.doi.org/10.1002/jbmr.3759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813857PMC
October 2019

Targeting vascular endothelial growth factor ameliorates PMMA-particles induced inflammatory osteolysis in murine calvaria.

Bone 2019 06 21;123:86-91. Epub 2019 Mar 21.

Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110, United States of America. Electronic address:

Cytokines and growth factors mediate inflammatory osteolysis in response to particles released from bone implants. However, the mechanism by which this process develops is not entirely clear. Blood vessels and related factors may be required to deliver immune cells and soluble factors to the injury site. Therefore, in the current study we investigated if, vascular endothelial growth factor (VEGF), which is required for angiogenesis, mediates polymethylmethacrylate (PMMA) particles-induced osteolysis. Using bone marrow derived macrophages (BMMs) and ST2 stromal cell line, we show that PMMA particles increase VEGF expression. Further, using a murine calvarial osteolysis model, we found that PMMA injection over calvaria induce significant increase in VEGF expression as well as new vessel formation, represented by von Willebrand factor (vWF) staining. Co-treatment using a VEGF-neutralizing antibody abrogated expression of vWF, indicating decreased angiogenesis. Finally, VEGF neutralizing antibody reduced expression of Tumor necrosis factor (TNF) and decreased osteoclastogenesis induced by PMMA particles in calvariae. This work highlights the significance of angiogenesis, specifically VEGF, as key driver of PMMA particle-induced inflammatory osteolysis, inhibition of which attenuates this response.
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http://dx.doi.org/10.1016/j.bone.2019.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491226PMC
June 2019

Targeting the NF-κB signaling pathway in chronic tendon disease.

Sci Transl Med 2019 02;11(481)

Department of Orthopedic Surgery, Columbia University, 650 W 168th St, New York, NY 10032, USA.

Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention; inflammation drives tendon degeneration before tearing and impairs healing after repair. Clinical evidence has implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as a correlate of pain-free return to function after surgical repair. However, it is currently unknown whether this response is a reaction to or a driver of pathology. Therefore, we aimed to understand the clinically relevant involvement of the NF-κB pathway in tendinopathy, to determine its potential causative roles in tendon degeneration, and to test its potential as a therapeutic candidate. Transcriptional profiling of early rotator cuff tendinopathy identified increases in NF-κB signaling, including increased expression of the regulatory serine kinase subunit IKKβ, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKβ in tendon fibroblasts, we observed degeneration of mouse rotator cuff tendons and the adjacent humeral head. These changes were associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, genetic deletion of IKKβ in tendon fibroblasts partially protected mice from chronic overuse-induced tendinopathy. Furthermore, conditional knockout of IKKβ improved outcomes after surgical repair, whereas overexpression impaired tendon healing. Accordingly, targeting of the IKKβ/NF-κB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders.
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http://dx.doi.org/10.1126/scitranslmed.aav4319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534967PMC
February 2019

Gasdermin D mediates the pathogenesis of neonatal-onset multisystem inflammatory disease in mice.

PLoS Biol 2018 11 2;16(11):e3000047. Epub 2018 Nov 2.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which-upon activation by the inflammasomes-translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice.
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http://dx.doi.org/10.1371/journal.pbio.3000047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235378PMC
November 2018

ASXL1 impairs osteoclast formation by epigenetic regulation of NFATc1.

Blood Adv 2018 10;2(19):2467-2477

Department of Pathology and Immunology.

Additional sex comb-like 1 () mutations are commonly associated with myeloid malignancies and are markers of aggressive disease. The fact that ASXL1 is necessary for myeloid differentiation raises the possibility it also regulates osteoclasts. We find deletion of ASXL1 in myeloid cells results in bone loss with increased abundance of osteoclasts. Because ASXL1 is an enhancer of trithorax and polycomb (ETP) protein, we asked if it modulates osteoclast differentiation by maintaining balance between positive and negative epigenetic regulators. In fact, loss of ASXL1 induces concordant loss of inhibitory H3K27me3 with gain of H3K4me3 at key osteoclast differentiation genes, including nuclear factor for activated T cells 1 () and In the setting of ASXL1 deficiency, increased NFATc1 binds to the () promoter thereby enhancing expression of this pro-osteoclastogenic gene. The global reduction of K27 trimethylation in ASXL1-deficient osteoclasts is also attended by a 40-fold increase in expression of the histone demethylase Jumonji domain-containing 3 (). Jmjd3 knockdown in ASXL1-deficient osteoclast precursors increases H3K27me3 on the promoter and impairs osteoclast formation. Thus, in addition to promoting myeloid malignancies, ASXL1 controls epigenetic reprogramming of osteoclasts to regulate bone resorption and mass.
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http://dx.doi.org/10.1182/bloodadvances.2018018309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177649PMC
October 2018

Selective inhibition of the p38α MAPK-MK2 axis inhibits inflammatory cues including inflammasome priming signals.

J Exp Med 2018 05 16;215(5):1315-1325. Epub 2018 Mar 16.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO

p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α-MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1β expression by promoting IL-1β mRNA degradation. Thus, IL-1β is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α-MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.
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http://dx.doi.org/10.1084/jem.20172063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940269PMC
May 2018

From the Cover: Lung-Specific Overexpression of Constitutively Active IKK2 Induces Pulmonary and Systemic Inflammations but Not Hypothalamic Inflammation and Glucose Intolerance.

Toxicol Sci 2017 Nov;160(1):4-14

Cardiology Division, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201.

The lung is constantly exposed to ambient pollutants such as ambient fine particulate matter (PM2.5), making it one of the most frequent locations of inflammation in the body. Given the establishment of crucial role of inflammation in the pathogenesis of cardiometabolic diseases, pulmonary inflammation is thus widely believed to be an important risk factor for cardiometabolic diseases. However, the causality between them has not yet been well established. To determine if pulmonary inflammation is sufficient to cause adverse cardiometabolic effects, SFTPC-rtTA+/-tetO-cre+/-pROSA-inhibitor κB kinase 2(IKK2)ca+/- (LungIKK2ca) and littermate SFTPC-rtTA+/-tetO-cre-/-pROSA-IKK2ca+/- wildtype (WT) mice were fed with doxycycline diet to induce constitutively active Ikk2 (Ikk2ca) overexpression in the lung and their pulmonary, systemic, adipose, and hypothalamic inflammations, vascular function, and glucose homeostasis were assessed. Feeding with doxycycline diet resulted in IKK2ca overexpression in the lungs of LungIKK2ca but not WT mice. This induction of IKK2ca was accompanied by marked pulmonary inflammation as evidenced by significant increases in bronchoalveolar lavage fluid leukocytes, pulmonary macrophage infiltration, and pulmonary mRNA expression of tumor necrosis factor α (Tnfα) and interleukin-6 (Il-6). This pulmonary inflammation due to lung-specific overexpression of IKK2ca was sufficient to increase circulating TNFα and IL-6 levels, adipose expression of Tnfα and Il-6 mRNA, aortic endothelial dysfunction, and systemic insulin resistance. Unexpectedly, no significant alteration in hypothalamic expression of Tnfα and Il-6 mRNA and glucose intolerance were observed in these mice. Pulmonary inflammation is sufficient to induce systemic inflammation, endothelial dysfunction, and insulin resistance, but not hypothalamic inflammation and glucose intolerance.
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http://dx.doi.org/10.1093/toxsci/kfx154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837620PMC
November 2017

Loss of Dnmt3b in Chondrocytes Leads to Delayed Endochondral Ossification and Fracture Repair.

J Bone Miner Res 2018 02 2;33(2):283-297. Epub 2017 Nov 2.

Department of Orthopaedic Surgery, School of Medicine, Washington University, St. Louis, MO, USA.

Despite advanced understanding of signaling mediated by local and systemic factors, the role of epigenetic factors in the regulation of bone regeneration remains vague. The DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b have tissue specific expression patterns and create unique methylation signatures to regulate gene expression. Using a stabilized murine tibia fracture model we find that Dnmt3b is induced early in fracture healing, peaks at 10 days post fracture (dpf), and declines to nearly undetectable levels by 28 dpf. Dnmt3b expression was cell-specific and stage-specific. High levels were observed in chondrogenic lineage cells within the fracture callus. To determine the role of Dnmt3b in fracture healing, Agc1Cre ;Dnmt3b (Dnmt3b ) mice were generated to delete Dnmt3b in chondrogenic cells. Dnmt3b fracture displayed chondrogenesis and chondrocyte maturation defect, and a delay in the later events of angiogenesis, ossification, and bone remodeling. Biomechanical studies demonstrated markedly reduced strength in Dnmt3b fractures and confirmed the delay in repair. The angiogenic response was reduced in both vessel number and volume at 10 and 14 dpf in Dnmt3b mice. Immunohistochemistry showed decreased CD31 expression, consistent with the reduced angiogenesis. Finally, in vitro angiogenesis assays with human umbilical vein endothelial cells (HUVECs) revealed that loss of Dnmt3b in chondrocytes significantly reduced tube formation and endothelial migration. To identify specific angiogenic factors involved in the decreased callus vascularization, a protein array was performed using conditioned media isolated from control and Dnmt3b loss-of-function chondrocytes. Several angiogenic factors, including CXCL12 and osteopontin (OPN) were reduced in chondrocytes following loss of Dnmt3b. DNA methylation analysis further identified hypomethylation in Cxcl12 promoter region. Importantly, the defects in tube formation and cell migration could be rescued by administration of CXCL12 and/or OPN. Altogether, our findings establish that Dnmt3b positively regulates chondrocyte maturation process, and its genetic ablation leads to delayed angiogenesis and fracture repair. © 2017 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809267PMC
February 2018

NUMBL Interacts with TAK1, TRAF6 and NEMO to Negatively Regulate NF-κB Signaling During Osteoclastogenesis.

Sci Rep 2017 10 3;7(1):12600. Epub 2017 Oct 3.

Department of Orthopaedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO, 63110, USA.

NF-κB signaling is essential for osteoclast differentiation and skeletal homeostasis. We have reported recently that NUMB-like (NUMBL) protein modulates osteoclastogenesis by down regulating NF-κB activation. Herein, we decipher the mechanism underlying this phenomenon. We found that whereas NUMBL mRNA expression decreases upon stimulation of wild type (WT) bone marrow macrophages (BMMs) with RANKL, TAK1 deficiency in these cells leads to increased NUMBL and decreased TRAF6 and NEMO expression. These changes were restored upon WT-TAK1 expression, but not with catalytically inactive TAK1-K63W, suggesting that TAK1 enzymatic activity is required for these events. Forced expression of NUMBL inhibits osteoclast differentiation and function as evident by reduction in all hallmarks of osteoclastogenesis. Conversely, NUMBL-null BMMs, show increased osteoclast differentiation and mRNA expression of osteoclast marker genes. Post-translationally, K48-linked poly-ubiquitination of NUMBL is diminished in TAK1-null BMMs compared to elevated K48-poly-ubiquitination in WT cells, indicating increased stability of NUMBL in TAK1-null conditions. Further, our studies show that NUMBL directly interacts with TRAF6 and NEMO, and induces their K48-poly-ubiquitination mediated proteasomal degradation. Collectively, our data suggest that NUMBL and TAK1 are reciprocally regulated and that NUMBL acts as an endogenous regulator of NF-κB signaling and osteoclastogenesis by targeting the TAK1-TRAF6-NEMO axis.
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http://dx.doi.org/10.1038/s41598-017-12707-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626749PMC
October 2017

Bone matrix components activate the NLRP3 inflammasome and promote osteoclast differentiation.

Sci Rep 2017 07 26;7(1):6630. Epub 2017 Jul 26.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO, 63110, United States.

The NLRP3 inflammasome senses a variety of signals referred to as danger associated molecular patterns (DAMPs), including those triggered by crystalline particulates or degradation products of extracellular matrix. Since some DAMPs confer tissue-specific activation of the inflammasomes, we tested the hypothesis that bone matrix components function as DAMPs for the NLRP3 inflammasome and regulate osteoclast differentiation. Indeed, bone particles cause exuberant osteoclastogenesis in the presence of RANKL, a response that correlates with NLRP3 abundance and the state of inflammasome activation. To determine the relevance of these findings to bone homeostasis, we studied the impact of Nlrp3 deficiency on bone using pre-clinical mouse models of high bone turnover, including estrogen deficiency and sustained exposure to parathyroid hormone or RANKL. Despite comparable baseline indices of bone mass, bone loss caused by hormonal or RANKL perturbations is significantly reduced in Nlrp3 deficient than in wild type mice. Consistent with the notion that osteolysis releases DAMPs from bone matrix, pharmacologic inhibition of bone resorption by zoledronate attenuates inflammasome activation in mice. Thus, signals originating from bone matrix activate the NLRP3 inflammasome in the osteoclast lineage, and may represent a bone-restricted positive feedback mechanism that amplifies bone resorption in pathologic conditions of accelerated bone turnover.
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http://dx.doi.org/10.1038/s41598-017-07014-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529467PMC
July 2017

Chronic inflammation triggered by the NLRP3 inflammasome in myeloid cells promotes growth plate dysplasia by mesenchymal cells.

Sci Rep 2017 07 7;7(1):4880. Epub 2017 Jul 7.

Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri, USA.

Skeletal complications are common features of neonatal-onset multisystem inflammatory disease (NOMID), a disorder caused by NLRP3-activating mutations. NOMID mice in which NLRP3 is activated globally exhibit several characteristics of the human disease, including systemic inflammation and cartilage dysplasia, but the mechanisms of skeletal manifestations remain unknown. In this study, we find that activation of NLRP3 in myeloid cells, but not mesenchymal cells triggers chronic inflammation, which ultimately, causes growth plate and epiphyseal dysplasia in mice. These responses are IL-1 signaling-dependent, but independent of PARP1, which also functions downstream of NLRP3 and regulates skeletal homeostasis. Mechanistically, inflammation causes severe anemia and hypoxia in the bone environment, yet down-regulates the HIF-1α pathway in chondrocytes, thereby promoting the demise of these cells. Thus, activation of NLRP3 in hematopoietic cells initiates IL-1β-driven paracrine cascades, which promote abnormal growth plate development in NOMID mice.
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http://dx.doi.org/10.1038/s41598-017-05033-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501802PMC
July 2017

PPAR-γ regulates pharmacological but not physiological or pathological osteoclast formation.

Nat Med 2016 11;22(11):1203-1205

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

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http://dx.doi.org/10.1038/nm.4208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179330PMC
November 2016

Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors.

Sci Rep 2016 07 20;6:29896. Epub 2016 Jul 20.

Department of Orthopaedic Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.

The transcription factor NF-κB is central to numerous physiologic processes including bone development, and its activation is controlled by IKKγ (also called NEMO), the regulatory subunit of IKK complex. NEMO is X-linked, and mutations in this gene result in Incontinentia Pigmenti in human hemizygous females. In mice, global deficiency causes embryonic lethality. In addition, certain point mutations in the NEMO (IKBKG) human gene manifest skeletal defects implicating NEMO in the regulation of bone homeostasis. To specifically investigate such role, we conditionally deleted Nemo from osteoclast and myeloid progenitors. Morphometric, histologic, and molecular analyses demonstrate that myeloid NEMO deletion causes osteopetrosis in mice. Mechanistically, NEMO deficiency hampered activation of IKK complex in osteoclast precursors, causing arrest of osteoclastogenesis and apoptosis. Interestingly, inhibiting apoptosis by genetic ablation of TNFr1 significantly increased cell survival, but failed to rescue osteoclastogenesis or reverse osteopetrosis. Based on this observation, we analyzed the expression of different regulators of osteoclastogenesis and discovered that NEMO deletion leads to increased RBPJ expression, resulting in a decrease of Blimp1 expression. Consequently, expression of IRF8 and Bcl6 which are targets of Blimp1 and potent osteoclastogenic transcriptional repressors, is increased. Thus, NEMO governs survival and osteoclast differentiation programs through serial regulation of multiple transcription factors.
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http://dx.doi.org/10.1038/srep29896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951754PMC
July 2016

Inflammation and epigenetic regulation in osteoarthritis.

Connect Tissue Res 2017 01 7;58(1):49-63. Epub 2016 Jul 7.

a Department of Orthopaedic Surgery , Washington University School of Medicine , St. Louis , MO , USA.

Osteoarthritis (OA) was once defined as a non-inflammatory arthropathy, but it is now well-recognized that there is a major inflammatory component to this disease. In addition to synovial cells, articular chondrocytes and other cells of diarthrodial joints are also known to express inflammatory mediators. It has been proposed that targeting inflammation pathways could be a promising strategy to treat OA. There have been many reports of cross-talk between inflammation and epigenetic factors in cartilage. Specifically, inflammatory mediators have been shown to regulate levels of enzymes that catalyze changes in DNA methylation and histone structure, as well as alter levels of non-coding RNAs. In addition, expression levels of a number of these epigenetic factors have been shown to be altered in OA, thereby suggesting potential interplay between inflammation and epigenetics in this disease. This review provides information on inflammatory pathways in arthritis and summarizes published research on how epigenetic regulators are affected by inflammation in chondrocytes. Furthermore, we discuss data showing how altered expression of some of these epigenetic factors can induce either catabolic or anti-catabolic effects in response to inflammatory signals. A better understanding of how inflammation affects epigenetic factors in OA may provide us with novel therapeutic strategies to treat this condition.
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http://dx.doi.org/10.1080/03008207.2016.1208655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266560PMC
January 2017

Regulation of NF-κB signaling in osteoclasts and myeloid progenitors.

Methods Mol Biol 2015 ;1280:527-42

Department of Orthopedic Surgery-Research, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8233, Saint Louis, MO, 63110, USA.

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is crucial for immune responses and skeletal development. Work in recent years has shown that various members of the NF-κB family are viable targets to regulate activity and survival of bone cells and hence bone metabolism. In this regard, deletion of upstream kinases or distal NF-κB subunits resulted with bone deformities. Thus, it has become increasingly apparent that detailed investigation of NF-κB in bone cells may provide opportunities to design new therapeutic modalities. In this chapter we present modified methodology describing efficient approaches to regulate the NF-κB pathway in vitro and in vivo to assess its function in bone cells and tissues.
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http://dx.doi.org/10.1007/978-1-4939-2422-6_31DOI Listing
November 2015

Osteopetrosis in TAK1-deficient mice owing to defective NF-κB and NOTCH signaling.

Proc Natl Acad Sci U S A 2015 Jan 22;112(1):154-9. Epub 2014 Dec 22.

Department of Orthopaedic Surgery and Cell Biology & Physiology and

The MAP kinase TGFβ-activated kinase (TAK1) plays a crucial role in physiologic and pathologic cellular functions including cell survival, differentiation, apoptosis, inflammation, and oncogenesis. However, the entire repertoire of its mechanism of action has not been elucidated. Here, we found that ablation of Tak1 in myeloid cells causes osteopetrosis in mice as a result of defective osteoclastogenesis. Mechanistically, Tak1 deficiency correlated with increased NUMB-like (NUMBL) levels. Accordingly, forced expression of Numbl abrogated osteoclastogenesis whereas its deletion partially restored osteoclastogenesis and reversed the phenotype of Tak1 deficiency. Tak1 deletion also down-regulated Notch intracellular domain (NICD), but increased the levels of the transcription factor recombinant recognition sequence binding protein at Jκ site (RBPJ), consistent with NUMBL regulating notch signaling through degradation of NICD, a modulator of RBPJ. Accordingly, deletion of Rbpj partially corrected osteopetrosis in Tak1-deficient mice. Furthermore, expression of active IKK2 in RBPJ/TAK1-deficient cells significantly restored osteoclastogenesis, indicating that activation of NF-κB is essential for complete rescue of the pathway. Thus, we propose that TAK1 regulates osteoclastogenesis by integrating activation of NF-κB and derepression of NOTCH/RBPJ in myeloid cells through inhibition of NUMBL.
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http://dx.doi.org/10.1073/pnas.1415213112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291677PMC
January 2015
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