PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

Abstract

Prostaglandin E2 (PGE2), a major cyclooxygenase-2 (COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis (OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts (EP4 ^LysM) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4 ^LysM mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of platelet-derived growth factor-BB (PDGF-BB) was also lower in the EP4 ^LysM mice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4 ^LysM mice. Finally, we showed that the Gαs/PI3K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

References

[1] Collaborators, G. B. D. R. F.. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet, 2018, 392: 1923-1994

[2] Peat, G. & Thomas, M. J. Osteoarthritis year in review 2020: epidemiology & therapy. Osteoarthr. Cartil. 29, 180–189 (2021).

[3] LiuQ, WangS, LinJ, ZhangY. The burden for knee osteoarthritis among Chinese elderly: estimates from a nationally representative study. Osteoarthr. Cartil., 2018, 26: 1636-1642

[4] RobinsonWH, et al.. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol., 2016, 12: 580-592

[5] MadryH. The subchondral bone: a new frontier in articular cartilage repair. Knee Surg. Sports Traumatol. Arthrosc., 2010, 18: 417-418

[6] GoldringSR. Alterations in periarticular bone and cross talk between subchondral bone and articular cartilage in osteoarthritis. Ther. Adv. Musculoskelet. Dis., 2012, 4: 249-258

[7] KloppenburgM, BerenbaumF. Osteoarthritis year in review 2019: epidemiology and therapy. Osteoarthr. Cartil., 2020, 28: 242-248

[8] NelsonAE. Osteoarthritis year in review 2017: clinical. Osteoarthr. Cartil., 2018, 26: 319-325

[9] FarnaghiS, CrawfordR, XiaoY, PrasadamI. Cholesterol metabolism in pathogenesis of osteoarthritis disease. Int. J. Rheum. Dis., 2017, 20: 131-140

[10] ChoiWS, et al.. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism regulates osteoarthritis. Nature, 2019, 566: 254-258

[11] ChangSH, et al.. Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-kappaB pathway. Nat. Commun., 2019, 10 ArticleID: 1442

[12] JimenezG, Cobo-MolinosJ, AntichC, Lopez-RuizE. Osteoarthritis: trauma vs disease. Adv. Exp. Med. Biol., 2018, 1059: 63-83

[13] PalazzoC, NguyenC, Lefevre-ColauMM, RannouF, PoiraudeauS. Risk factors and burden of osteoarthritis. Ann. Phys. Rehab. Med., 2016, 59: 134-138

[14] CuiZ, et al.. Halofuginone attenuates osteoarthritis by inhibition of TGF-beta activity and H-type vessel formation in subchondral bone. Ann. Rheum. Dis., 2016, 75: 1714-1721

[15] ZhenG, et al.. Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat. Med., 2013, 19: 704-712

[16] Su, W. et al. Angiogenesis stimulated by elevated PDGF-BB in subchondral bone contributes to osteoarthritis development. JCI Insight 5, e135446 (2020).

[17] LinC, et al.. Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12. Bone Res., 2019, 7: 5

[18] LoriesRJ, LuytenFP. The bone-cartilage unit in osteoarthritis. Nat. Rev. Rheumatol., 2011, 7: 43-49

[19] CinqueME, DornanGJ, ChahlaJ, MoatsheG, LaPradeRF. High rates of osteoarthritis develop after anterior cruciate ligament surgery: an analysis of 4108 patients. Am. J. Sports Med., 2018, 46: 2011-2019

[20] ThijssenE, van CaamA, van der KraanPM. Obesity and osteoarthritis, more than just wear and tear: pivotal roles for inflamed adipose tissue and dyslipidaemia in obesity-induced osteoarthritis. Rheumatology, 2015, 54: 588-600

[21] de ZwartAH, et al.. Factors associated with upper leg muscle strength in knee osteoarthritis: a scoping review. J. Rehab. Med., 2018, 50: 140-150

[22] LiG, et al.. Influence of age and gender on microarchitecture and bone remodeling in subchondral bone of the osteoarthritic femoral head. Bone, 2015, 77: 91-97

[23] RoemerFW, et al.. Change in MRI-detected subchondral bone marrow lesions is associated with cartilage loss: the MOST Study. A longitudinal multicentre study of knee osteoarthritis. Ann. Rheum. Dis., 2009, 68: 1461-1465

[24] HunterDJ, et al.. Increase in bone marrow lesions associated with cartilage loss: a longitudinal magnetic resonance imaging study of knee osteoarthritis. Arthritis Rheumat., 2006, 54: 1529-1535

[25] RaynauldJP, et al.. Correlation between bone lesion changes and cartilage volume loss in patients with osteoarthritis of the knee as assessed by quantitative magnetic resonance imaging over a 24-month period. Ann. Rheum. Dis., 2008, 67: 683-688

[26] RaggattLJ, PartridgeNC. Cellular and molecular mechanisms of bone remodeling. J. Biol. Chem., 2010, 285: 25103-25108

[27] LacourtM, et al.. Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis. Osteoarthr. Cartil., 2012, 20: 572-583

[28] ZhenG, CaoX. Targeting TGFbeta signaling in subchondral bone and articular cartilage homeostasis. Trends Pharmacol. Sci., 2014, 35: 227-236

[29] PengY, WuS, LiY, CraneJL. Type H blood vessels in bone modeling and remodeling. Theranostics, 2020, 10: 426-436

[30] Hu, W., Chen, Y., Dou, C. & Dong, S. Microenvironment in subchondral bone: predominant regulator for the treatment of osteoarthritis. Ann. Rheum. Dis. (2020).

[31] ZhuS, et al.. Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain. J. Clin. Investig., 2019, 129: 1076-1093

[32] NwosuLN, MappPI, ChapmanV, WalshDA. Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis. Ann. Rheum. Dis., 2016, 75: 1246-1254

[33] LaslettLL, et al.. Zoledronic acid reduces knee pain and bone marrow lesions over 1 year: a randomised controlled trial. Ann. Rheum. Dis., 2012, 71: 1322-1328

[34] BallalP, et al.. The relation of oral bisphosphonates to bone marrow lesion volume among women with osteoarthritis. Osteoarthr. Cartil., 2020, 28: 1325-1329

[35] VarennaM, ZucchiF, FailoniS, BeccioliniA, BerrutoM. Intravenous neridronate in the treatment of acute painful knee osteoarthritis: a randomized controlled study. Rheumatology, 2015, 54: 1826-1832

[36] RossiniM, et al.. Effects of intra-articular clodronate in the treatment of knee osteoarthritis: results of a double-blind, randomized placebo-controlled trial. Rheumatol. Int., 2015, 35: 255-263

[37] VaysbrotEE, OsaniMC, MusettiMC, McAlindonTE, BannuruRR. Are bisphosphonates efficacious in knee osteoarthritis? A meta-analysis of randomized controlled trials. Osteoarthr. Cartil., 2018, 26: 154-164

[38] EriksenEF, ShabestariM, GhouriA, ConaghanPG. Bisphosphonates as a treatment modality in osteoarthritis. Bone, 2021, 143: 115352

[39] ZhangY, DaakaY. PGE2 promotes angiogenesis through EP4 and PKA Cgamma pathway. Blood, 2011, 118: 5355-5364

[40] Lu, W. et al. Reprogramming immunosuppressive myeloid cells facilitates immunotherapy for colorectal cancer. EMBO Mol. Med. 13, e12798 (2020).

[41] NiS, et al.. Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice. Nat. Commun., 2019, 10 ArticleID: 5643

[42] NakanishiM, RosenbergDW. Multifaceted roles of PGE2 in inflammation and cancer. Semin. Immunopathol., 2013, 35: 123-137

[43] JinJ, et al.. Prostaglandin E2 regulates renal function in C57/BL6 mouse with 5/6 nephrectomy. Life Sci., 2017, 174: 68-76

[44] TuM, et al.. Inhibition of cyclooxygenase-2 activity in subchondral bone modifies a subtype of osteoarthritis. Bone Res., 2019, 7: 29

[45] AminAR, et al.. Superinduction of cyclooxygenase-2 activity in human osteoarthritis-affected cartilage. Influence of nitric oxide. J. Clin. Investig., 1997, 99: 1231-1237

[46] SugimotoY, NarumiyaS. Prostaglandin E receptors. J. Biol. Chem., 2007, 282: 11613-11617

[47] SatoT, et al.. Prostaglandin EP2 receptor signalling inhibits the expression of matrix metalloproteinase 13 in human osteoarthritic chondrocytes. Ann. Rheum. Dis., 2011, 70: 221-226

[48] WeinrebM, et al.. Expression of the prostaglandin E(2) (PGE(2) receptor subtype EP(4) and its regulation by PGE(2) in osteoblastic cell lines and adult rat. Bone Tissue Bone, 2001, 28: 275-281

[49] YoshidaK, et al.. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc. Natl. Acad. Sci. USA, 2002, 99: 4580-4585

[50] AtturM, et al.. Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor. J. Immunol., 2008, 181: 5082-5088

[51] NishitaniK, et al.. PGE2 inhibits MMP expression by suppressing MKK4-JNK MAP kinase-c-JUN pathway via EP4 in human articular chondrocytes. J. Cell. Biochem., 2010, 109: 425-433

[52] AshrafS, et al.. Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis. Ann. Rheum. Dis., 2014, 73: 1710-1718

[53] HunterDJ, Bierma-ZeinstraS. Osteoarthritis. Lancet, 2019, 393: 1745-1759

[54] Tombran-TinkJ, BarnstableCJ. Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone. Biochem. Biophys. Res. Commun., 2004, 316: 573-579

[55] KimBJ, et al.. Osteoclast-secreted SLIT3 coordinates bone resorption and formation. J. Clin. Investig., 2018, 128: 1429-1441

[56] LiuX, et al.. Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis. Nat. Commun., 2021, 12 ArticleID: 1832

[57] YangJJ, et al.. Discovery and characterization of 1H-1,2,3-triazole derivatives as novel prostanoid EP4 receptor antagonists for cancer immunotherapy. J. Med. Chem., 2020, 63: 569-590

[58] RaneMA, GitinA, FiedlerB, FiedlerL, HennekensCH. Risks of cardiovascular disease and beyond in prescription of nonsteroidal anti-inflammatory drugs. J. Cardiovasc. Pharmacol. Ther., 2020, 25: 3-6

[59] WalkerC, BiasucciLM. Cardiovascular safety of non-steroidal anti-inflammatory drugs revisited. Postgrad. Med., 2018, 130: 55-71

[60] HawkeyCJ. COX-1 and COX-2 inhibitors. Best. Pract. Res. Clin. Gastroenterol., 2001, 15: 801-820

[61] Bjarnason, I. Gastrointestinal safety of NSAIDs and over-the-counter analgesics. Int. J. Clin. Pract. Supplement, 37-42, (2013).

[62] GaoM, et al.. Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts. Proc. Natl Acad. Sci. USA, 2015, 112: 8397-8402

[63] JohnstonJE. The best medicine sometimes can’t be bought. J. Miss. State Med. Assoc., 1992, 33: 97-98

[64] YokoyamaU, IwatsuboK, UmemuraM, FujitaT, IshikawaY. The prostanoid EP4 receptor and its signaling pathway. Pharmacol. Rev., 2013, 65: 1010-1052

[65] LiDZ, ZhangQX, DongXX, LiHD, MaX. Treatment with hydrogen molecules prevents RANKL-induced osteoclast differentiation associated with inhibition of ROS formation and inactivation of MAPK, AKT and NF-kappa B pathways in murine RAW264.7 cells. J. Bone Miner. Metab., 2014, 32: 494-504

[66] MendozaMC, ErEE, BlenisJ. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem. Sci., 2011, 36: 320-328

[67] Kalbasi AnarakiP, et al.. Urokinase receptor mediates osteoclastogenesis via M-CSF release from osteoblasts and the c-Fms/PI3K/Akt/NF-kappaB pathway in osteoclasts. J. Bone Miner. Res., 2015, 30: 379-388

[68] HayamiT, et al.. The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheumat., 2004, 50: 1193-1206

[69] KobayashiY, et al.. Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1. J. Biol. Chem., 2005, 280: 11395-11403

[70] ManoM, et al.. Prostaglandin E2 directly inhibits bone-resorbing activity of isolated mature osteoclasts mainly through the EP4 receptor. Calcif. Tissue Int., 2000, 67: 85-92

[71] LiX, et al.. Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritis. Arthritis Rheumat., 2009, 60: 513-523

[72] OnoK, et al.. Biphasic effect of prostaglandin E2 on osteoclast formation in spleen cell cultures: role of the EP2 receptor. J. Bone Miner. Res., 2005, 20: 23-29

[73] GilmanKE, LimesandKH. The complex role of prostaglandin E2-EP receptor signaling in wound healing. Am. J. Physiol. Regulatory, Integr. Comp. Physiol., 2021, 320: R287-R296

[74] DieppePA, LohmanderLS. Pathogenesis and management of pain in osteoarthritis. Lancet, 2005, 365: 965-973

[75] WalshDA, et al.. Angiogenesis and nerve growth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis. Rheumatology, 2010, 49: 1852-1861

[76] LuJ, et al.. Positive-feedback regulation of subchondral H-type vessel formation by chondrocyte promotes osteoarthritis development in mice. J. Bone Miner. Res., 2018, 33: 909-920

[77] XieH, et al.. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nat. Med., 2014, 20: 1270-1278

[78] PuljakL, et al.. Celecoxib for osteoarthritis. Cochrane Database Syst. Rev., 2017, 5: CD009865

[79] AngeliF, TrapassoM, SignorottiS, VerdecchiaP, ReboldiG. Amlodipine and celecoxib for treatment of hypertension and osteoarthritis pain. Expert Rev. Clin. Pharmacol., 2018, 11: 1073-1084

[80] ClemettD, GoaKL. Celecoxib: a review of its use in osteoarthritis, rheumatoid arthritis and acute pain. Drugs, 2000, 59: 957-980

[81] LiG, et al.. Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes. Arthritis Res. Ther., 2013, 15: 223

[82] AaronRK, RacineJR, VoisinetA, EvangelistaP, DykeJP. Subchondral bone circulation in osteoarthritis of the human knee. Osteoarthr. Cartil., 2018, 26: 940-944

[83] BurrDB, GallantMA. Bone remodelling in osteoarthritis. Nat. Rev. Rheumatol., 2012, 8: 665-673

[84] MansellJP, CollinsC, BaileyAJ. Bone, not cartilage, should be the major focus in osteoarthritis. Nat. Clin. Pract. Rheumatol., 2007, 3: 306-307

[85] Martel-PelletierJ, et al.. Osteoarthritis. Nat. Rev. Dis. Prim., 2016, 2: 16072

[86] SpectorTD, et al.. Effect of risedronate on joint structure and symptoms of knee osteoarthritis: results of the BRISK randomized, controlled trial [ISRCTN01928173]. Arthritis Res. Ther., 2005, 7: R625-R633

[87] BinghamCO 3rd, et al.. Risedronate decreases biochemical markers of cartilage degradation but does not decrease symptoms or slow radiographic progression in patients with medial compartment osteoarthritis of the knee: results of the two-year multinational knee osteoarthritis structural arthritis study. Arthritis Rheumat., 2006, 54: 3494-3507

[88] YueR, et al.. Beta-arrestin1 regulates zebrafish hematopoiesis through binding to YY1 and relieving polycomb group repression. Cell, 2009, 139: 535-546

[89] LuoJ, et al.. LGR4 is a receptor for RANKL and negatively regulates osteoclast differentiation and bone resorption. Nat. Med., 2016, 22: 539-546

[90] PritzkerKP, et al.. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthr. Cartil., 2006, 14: 13-29

[91] MoriH, CardiffRD. Methods of immunohistochemistry and immunofluorescence: converting invisible to visible. Methods Mol. Biol., 2016, 1458: 1-12

[92] FengJ, et al.. Blocking STAT3 by pyrvinium pamoate causes metabolic lethality in KRAS-mutant lung cancer. Biochem. Pharmacol., 2020, 177: 113960

Funding

National Natural Science Foundation of China (National Science Foundation of China)(91949127)

National Key Research and Development Program of China (2020YFC2002800),National Key Research and Development Program of China (2018YFC1105102),Fundamental Research Funds for the Central Universities (22120210586).

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