Inhibition of fibroblast activation protein ameliorates cartilage matrix degradation and osteoarthritis progression

Abstract

Fibroblast activation protein (Fap) is a serine protease that degrades denatured type I collagen, α2-antiplasmin and FGF21. Fap is highly expressed in bone marrow stromal cells and functions as an osteogenic suppressor and can be inhibited by the bone growth factor Osteolectin (Oln). Fap is also expressed in synovial fibroblasts and positively correlated with the severity of rheumatoid arthritis (RA). However, whether Fap plays a critical role in osteoarthritis (OA) remains poorly understood. Here, we found that Fap is significantly elevated in osteoarthritic synovium, while the genetic deletion or pharmacological inhibition of Fap significantly ameliorated posttraumatic OA in mice. Mechanistically, we found that Fap degrades denatured type II collagen (Col II) and Mmp13-cleaved native Col II. Intra-articular injection of rFap significantly accelerated Col II degradation and OA progression. In contrast, Oln is expressed in the superficial layer of articular cartilage and is significantly downregulated in OA. Genetic deletion of Oln significantly exacerbated OA progression, which was partially rescued by Fap deletion or inhibition. Intra-articular injection of rOln significantly ameliorated OA progression. Taken together, these findings identify Fap as a critical pathogenic factor in OA that could be targeted by both synthetic and endogenous inhibitors to ameliorate articular cartilage degradation.

References

[1] Safiri S et al. Global, regional and national burden of osteoarthritis 1990–2017: a systematic analysis of the global burden of disease study 2017. Ann. Rheum. Dis., 2020, 79: 819-828

[2] Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet (Lond., Engl.), 2011, 377: 2115-2126

[3] Turkiewicz A et al. Current and future impact of osteoarthritis on health care: a population-based study with projections to year 2032. Osteoarthr. Cartil., 2014, 22: 1826-1832

[4] Wei Y, Bai L. Recent advances in the understanding of molecular mechanisms of cartilage degeneration, synovitis and subchondral bone changes in osteoarthritis. Connect. Tissue Res., 2016, 57: 245-261

[5] van der Kraan PM, van den Berg WB. Osteophytes: relevance and biology. Osteoarthr. Cartil., 2007, 15: 237-244

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

[7] Hu Y, Chen X, Wang S, Jing Y, Su J. Subchondral bone microenvironment in osteoarthritis and pain. Bone Res., 2021, 9: 20

[8] Zhen G, Cao X. Targeting TGFbeta signaling in subchondral bone and articular cartilage homeostasis. Trends Pharm. Sci., 2014, 35: 227-236

[9] Pap T, Korb-Pap A. Cartilage damage in osteoarthritis and rheumatoid arthritis-two unequal siblings. Nat. Rev. Rheumatol., 2015, 11: 606-615

[10] Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat. Rev. Rheumatol., 2011, 7: 33-42

[11] Troeberg L, Nagase H. Proteases involved in cartilage matrix degradation in osteoarthritis. Biochim. Biophys. Acta, 2012, 1824: 133-145

[12] Gracitelli GC, Moraes VY, Franciozi CE, Luzo MV, Belloti JC. Surgical interventions (microfracture, drilling, mosaicplasty, and allograft transplantation) for treating isolated cartilage defects of the knee in adults. Cochrane Database Syst. Rev., 2016, 9: Cd010675

[13] Angele P et al. Chondral and osteochondral operative treatment in early osteoarthritis. Knee Surg. Sports Traumatol. Arthrosc., 2016, 24: 1743-1752

[14] Glyn-Jones S et al. Osteoarthritis. Lancet (Lond., Engl.), 2015, 386: 376-387

[15] Kolasinski SL et al. 2019 American College of Rheumatology/Arthritis Foundation guideline for the management of osteoarthritis of the hand, hip, and knee. Arthritis Rheumatol., 2020, 72: 220-233

[16] Bannuru RR et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthr. Cartil., 2019, 27: 1578-1589

[17] Bruyère O et al. An updated algorithm recommendation for the management of knee osteoarthritis from the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO). Semin. Arthritis Rheum., 2019, 49: 337-350

[18] Filardo G et al. Non-surgical treatments for the management of early osteoarthritis. Knee Surg. Sports Traumatol. Arthrosc., 2016, 24: 1775-1785

[19] Heinegard D, Saxne T. The role of the cartilage matrix in osteoarthritis. Nat. Rev. Rheumatol., 2011, 7: 50-56

[20] Bondeson J, Wainwright S, Hughes C, Caterson B. The regulation of the ADAMTS4 and ADAMTS5 aggrecanases in osteoarthritis: a review. Clin. Exp. Rheumatol., 2008, 26: 139-145

[21] Mehana EE, Khafaga AF, El-Blehi SS. The role of matrix metalloproteinases in osteoarthritis pathogenesis: an updated review. Life Sci., 2019, 234: 116786

[22] Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J. Biol. Chem., 1996, 271: 1544-1550

[23] Wan Y et al. Selective MMP-13 inhibitors: promising agents for the therapy of osteoarthritis. Curr. Med Chem., 2020, 27: 3753-3769

[24] Mort JS, Billington CJ. Articular cartilage and changes in arthritis: matrix degradation. Arthritis Res., 2001, 3: 337-341

[25] Mitchell PG et al. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J. Clin. Investig., 1996, 97: 761-768

[26] Bányai L, Tordai H, Patthy L. The gelatin-binding site of human 72 kDa type IV collagenase (gelatinase A). Biochem. J., 1994, 298: 403-407

[27] Overall CM, Sodek J. Initial characterization of a neutral metalloproteinase, active on native 3/4-collagen fragments, synthesized by ROS 17/2.8 osteoblastic cells, periodontal fibroblasts, and identified in gingival crevicular fluid. J. Dent. Res., 1987, 66: 1271-1282

[28] Renkiewicz R et al. Broad-spectrum matrix metalloproteinase inhibitor marimastat-induced musculoskeletal side effects in rats. Arthritis Rheum., 2003, 48: 1742-1749

[29] Krzeski P et al. Development of musculoskeletal toxicity without clear benefit after administration of PG-116800, a matrix metalloproteinase inhibitor, to patients with knee osteoarthritis: a randomized, 12-month, double-blind, placebo-controlled study. Arthritis Res. Ther., 2007, 9: R109

[30] Milner JM, Patel A, Rowan AD. Emerging roles of serine proteinases in tissue turnover in arthritis. Arthritis Rheum., 2008, 58: 3644-3656

[31] Chou PY, Su CM, Huang CY, Tang CH. The characteristics of thrombin in osteoarthritic pathogenesis and treatment. Biomed. Res. Int., 2014, 2014: 407518

[32] Wilkinson DJ et al. Matriptase induction of metalloproteinase-dependent aggrecanolysis in vitro and in vivo: promotion of osteoarthritic cartilage damage by multiple mechanisms. Arthritis Rheumatol., 2017, 69: 1601-1611

[33] Akhatib B et al. Chondroadherin fragmentation mediated by the protease HTRA1 distinguishes human intervertebral disc degeneration from normal aging. J. Biol. Chem., 2013, 288: 19280-19287

[34] Goldstein LA et al. Molecular cloning of seprase: a serine integral membrane protease from human melanoma. Biochim. Biophys. Acta, 1997, 1361: 11-19

[35] Park JE et al. Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J. Biol. Chem., 1999, 274: 36505-36512

[36] Balaziova E et al. Dipeptidyl peptidase-IV activity and/or structure homologs (DASH): contributing factors in the pathogenesis of rheumatic diseases? Adv. Exp. Med. Biol., 2006, 575: 169-174

[37] Hamson EJ, Keane FM, Tholen S, Schilling O, Gorrell MD. Understanding fibroblast activation protein (FAP): substrates, activities, expression and targeting for cancer therapy. Proteom. Clin. Appl., 2014, 8: 454-463

[38] Dunshee DR et al. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J. Biol. Chem., 2016, 291: 5986-5996

[39] Lee KN et al. Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein. Blood, 2006, 107: 1397-1404

[40] Keane FM, Nadvi NA, Yao TW, Gorrell MD. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-alpha. FEBS J., 2011, 278: 1316-1332

[41] Zhang HE et al. Identification of novel natural substrates of fibroblast activation protein-alpha by differential degradomics and proteomics. Mol. Cell Proteom., 2019, 18: 65-85

[42] Scanlan MJ et al. Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc. Natl. Acad. Sci. USA, 1994, 91: 5657-5661

[43] Rettig WJ et al. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc. Natl Acad. Sci. USA, 1988, 85: 3110-3114

[44] Kelly T. Fibroblast activation protein-alpha and dipeptidyl peptidase IV (CD26): cell-surface proteases that activate cell signaling and are potential targets for cancer therapy. Drug Resist. Updat., 2005, 8: 51-58

[45] Garin-Chesa P, Old LJ, Rettig WJ. Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc. Natl. Acad. Sci. USA, 1990, 87: 7235-7239

[46] Cortez E, Roswall P, Pietras K. Functional subsets of mesenchymal cell types in the tumor microenvironment. Semin. Cancer Biol., 2014, 25: 3-9

[47] Kou X et al. The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing. Sci. Transl. Med., 2018, 10: eaai8524

[48] Wei H et al. Identification of fibroblast activation protein as an osteogenic suppressor and anti-osteoporosis drug target. Cell Rep., 2020, 33: 108252

[49] Bauer S et al. Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes. Arthritis Res. Ther., 2006, 8: R171

[50] Waldele S et al. Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis. Arthritis Res. Ther., 2015, 17: 12

[51] Hiraoka A et al. Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells. Proc. Natl. Acad. Sci. USA, 1997, 94: 7577-7582

[52] Hiraoka A et al. Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells. Hematol. J., 2001, 2: 307-315

[53] Yue R, Shen B, Morrison SJ. Clec11a/osteolectin is an osteogenic growth factor that promotes the maintenance of the adult skeleton. eLife, 2016, 5: e18782

[54] Shen B et al. Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass. eLife, 2019, 8: e42274

[55] Edosada CY et al. Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity. J. Biol. Chem., 2006, 281: 7437-7444

[56] Niedermeyer J et al. Expression of the fibroblast activation protein during mouse embryo development. Int. J. Dev. Biol., 2001, 45: 445-447

[57] Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat. Protoc., 2008, 3: 1253-1260

[58] Goldring MB, Berenbaum F. Human chondrocyte culture models for studying cyclooxygenase expression and prostaglandin regulation of collagen gene expression. Osteoarthr. Cartil., 1999, 7: 386-388

[59] Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol., 2002, 21: 251-262

[60] Robbins JR et al. Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1beta. Arthritis Rheum., 2000, 43: 2189-2201

[61] Lee HO et al. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells. BMC Cancer, 2011, 11

[62] Nagaraju CK et al. Global fibroblast activation throughout the left ventricle but localized fibrosis after myocardial infarction. Sci. Rep., 2017, 7

[63] Brokopp CE et al. Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata. Eur. Heart J., 2011, 32: 2713-2722

[64] Christiansen VJ, Jackson KW, Lee KN, McKee PA. Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV. Arch. Biochem. Biophys., 2007, 457: 177-186

[65] Fan MH et al. Fibroblast activation protein (FAP) accelerates collagen degradation and clearance from lungs in mice. J. Biol. Chem., 2016, 291: 8070-8089

[66] Kumagai K et al. Changes of synovial fluid biomarker levels after opening wedge high tibial osteotomy in patients with knee osteoarthritis. Osteoarthr. Cartil., 2021, 29: 1020-1028

[67] Li L et al. Profiling of inflammatory mediators in the synovial fluid related to pain in knee osteoarthritis. BMC Musculoskelet. Disord., 2020, 21

[68] Van Spil WE, Kubassova O, Boesen M, Bay-Jensen AC, Mobasheri A. Osteoarthritis phenotypes and novel therapeutic targets. Biochem. Pharm., 2019, 165: 41-48

[69] Pineiro-Sanchez ML et al. Identification of the 170-kDa melanoma membrane-bound gelatinase (seprase) as a serine integral membrane protease. J. Biol. Chem., 1997, 272: 7595-7601

[70] Aertgeerts K et al. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J. Biol. Chem., 2005, 280: 19441-19444

[71] Deacon CF. Dipeptidyl peptidase 4 inhibitors in the treatment of type 2 diabetes mellitus. Nat. Rev. Endocrinol., 2020, 16: 642-653

[72] Croft AP et al. Distinct fibroblast subsets drive inflammation and damage in arthritis. Nature, 2019, 570: 246-251

[73] Metsäranta M et al. Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain. J. Cell Biol., 1992, 118: 203-212

[74] Rintala M, Metsäranta M, Säämänen AM, Vuorio E, Rönning O. Abnormal craniofacial growth and early mandibular osteoarthritis in mice harbouring a mutant type II collagen transgene. J. Anat., 1997, 190: 201-208

[75] Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front. Biosci., 2006, 11: 529-543

[76] Liu J, Khalil RA. Matrix metalloproteinase inhibitors as investigational and therapeutic tools in unrestrained tissue remodeling and pathological disorders. Prog. Mol. Biol. Transl. Sci., 2017, 148: 355-420

[77] Hollander AP et al. Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration. J. Clin. Investig., 1995, 96: 2859-2869

[78] Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann. Rheum. Dis., 1957, 16: 494-502

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

[80] Krenn V et al. Synovitis score: discrimination between chronic low-grade and high-grade synovitis. Histopathology, 2006, 49: 358-364

[81] Lorenz J, Grässel S. Experimental osteoarthritis models in mice. Methods Mol. Biol., 2014, 1194: 401-419

[82] Yao Z et al. Reduced PDGF-AA in subchondral bone leads to articular cartilage degeneration after strenuous running. J. Cell. Physiol., 2019, 234: 17946-17958

Funding

National Natural Science Foundation of China (National Science Foundation of China)(91749124, 81772389, 82070108)

the National Key R&D Program on Stem Cell and Translational Research (2017YFA0106400, 2021YFA1100900), the Fundamental Research Funds for the Central Universities (22120190149 and kx0200020173386)

Ministry of Science and Technology of the People’s Republic of China (Chinese Ministry of Science and Technology)(2020YFC2002804)

the Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (ZJ2018-ZD-004), the Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai.