Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid This information is current as Arthritis of October 2, 2021. Martine Charbonneau, Roxane R. Lavoie, Annie Lauzier, Kelly Harper, Patrick P. McDonald and Claire M. Dubois J Immunol 2016; 196:3264-3275; Prepublished online 14 March 2016; Downloaded from doi: 10.4049/jimmunol.1500502 http://www.jimmunol.org/content/196/8/3264 References This article cites 93 articles, 21 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/196/8/3264.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis Martine Charbonneau,*,1 Roxane R. Lavoie,*,1 Annie Lauzier,* Kelly Harper,* Patrick P. McDonald,† and Claire M. Dubois* Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-b that triggers formation of matrix-degrading invadosomes through mo- lecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-b in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-ab was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-ab was also elevated in RA Downloaded from synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A–D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-b upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-b neutralization or interference with the Smad/TbR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Ka was selectively involved in PDGF-B expression, http://www.jimmunol.org/ whereas both PI3Ka and PI3Kd participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-b and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-b. The Journal of Immunology, 2016, 196: 3264–3275. heumatoid arthritis (RA) is a systemic autoimmune dis- inflammation and joint destruction (2–4). Arthritic FLS resemble ease that mainly affects the joints, leading to joint in- transformed mesenchymal cells that are highly invasive in vitro R flammation and erosive structural damages. Although and in vivo. This property correlates with elevated production of by guest on October 2, 2021 important progress has been made in managing the pain and in- inflammatory cytokines and proteolytic enzymes that sustain in- flammation associated with the disease, strategies to directly in- flammation and joint matrix degradation. We have reported that terfere with the process of erosion are lacking. The onset of RA the ability of arthritic FLS to degrade the extracellular matrix causes important morphological changes in joint lining, including depends on the formation of plasma membrane structures that formation of an aggressive tumor-like synovial tissue that invades resembled invadopodia in tumor cells (5, 6). These structures were and erodes cartilage and bone (1). A large body of evidence from detected in fibroblast-like cells strategically located at the carti- patients and experimental animal models indicated that fibroblast- lage–synovial membrane interface. They were shown to contain like synoviocytes (FLS) are the main cell type that actively drives actin components, signaling molecules, such as Src, and high levels of proteolytic enzymes known to be particularly efficient at *Immunology Division, Department of Pediatrics, Faculty of Medicine, University of inducing cartilage damage. Importantly, interference with the Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada; and †Pneumology Division, Department of Medicine, Faculty of Medicine, University of Sherbrooke, Sherbrooke formation of invadosomes in arthritic FLS strongly inhibited Quebec J1H 5N4, Canada matrix degradation in vitro and ex vivo as well as cartilage deg- 1M.C. and R.R.L. are cofirst authors. radation in a rat model of arthritis (5, 6). These observations ORCIDs: 0000-0001-6253-8090 (R.R.L.); 0000-0002-0751-2821 (C.M.D.). suggested that invadosomes were directly involved in joint deg- Received for publication March 4, 2015. Accepted for publication February 15, 2016. radation, leading to the conclusion that an in-depth understanding This work was supported by Canadian Institutes for Health Research (CIHR) Grants of the mechanism of invadosome formation is of importance for MOP-86634 and MOP-286621 (to C.M.D.). C.M.D. is a member of the Fonds de la development of joint protection strategies for the clinical man- Recherche en Sante´ du Que´bec–funded Centre de Recherche du Centre Hospitalier agement of RA. Universitaire de Sherbrooke. K.H. is recipient of a scholarship from CIHR. The mechanisms involved in invadosome formation in synovial Address correspondence and reprint requests to Dr. Claire M. Dubois, Immunology Division, Department of Pediatrics, Faculty of Medicine, Universite´ de Sherbrooke, cells are not fully known. Invadosome formation and in vivo 3001 12th Avenue North, Sherbrooke, QC J1H 5N4, Canada. E-mail address: Claire. cartilage degradation capability of synovial cells of collagen- [email protected] induced arthritis rats were shown to depend on an autocrine ac- Abbreviations used in this article: ACR, American College of Rheumatology; tivation loop that involved TGF-b (6). Analysis of the protein and ECM, extracellular matrix; FLS, fibroblast-like synoviocyte; LPA, lysophosphatidic acid; NA, nonarthritis; OA, osteoarthritis; PDGF, platelet-derived growth factor; mRNA in RA synovial tissues revealed that TGF-b was highly PDGFR, PDGF receptor; PLC, phospholipase C; pY, phosphotyrosine; RA, rheumatoid expressed in RA patients (7–9). However, few studies have arthritis; RTK, receptor tyrosine kinase. addressed the role of TGF-b in the functions of synovial fibro- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 blasts derived from these patients. TGF-b was shown to increase www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500502 The Journal of Immunology 3265 the expression of proinflammatory cytokines and metalloproteinases agnosed using the 1986 ACR clinical criteria (34). The protocol was ap- in RA synoviocytes (9), an effect that was found to be dramati- proved by the Centre Hospitalier Universitaire de Sherbrooke Ethics cally potentiated by receptor tyrosine kinase (RTK)–dependent Committee, and written consent was obtained from all participants. Synoviocytes were isolated using standard procedures (35), and the signaling (10). These studies suggested a potential association culture was maintained in DMEM-F12 medium supplemented with 10% between TGF-b and RTK signaling to promote proarthritic FBS and 40 mg/ml gentamicin. Cells were used between passages 3 and 8. functions of synovial fibroblasts. Synoviocyte cultures exhibited a classic spindle-shape fibroblastic mor- RTKs comprise a large family of cell surface receptors that are phology that formed parallel clusters at confluency. The cell surface phenotypic marker analysis showed that they were consistently positive for essential components of signal transduction pathways that medi- the stromal mesenchymal marker fibronectin (.99%) and negative (,1%) ate cell survival, proliferation, differentiation, and motility, and for the macrophage marker CD68. modulate cell metabolism (11). These transmembrane proteins bind polypeptide ligands, mainly growth factors. Among the 58 Plasmids and transfections RTK family members, epidermal growth factor receptor, platelet- pLKO.1-puro short hairpin RNA targeting PI3Ka, PI3Kb, PI3Kd, and derived growth factor (PDGF) receptor (PDGFR), fibroblast control (scrambled) short hairpin RNA plasmids were from Sigma-Aldrich growth factor