http://hdl.handle.net/1765/109466 Identification of TGFβ-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets Laurie M.G. de Kroon1,2,&, Guus G.H. van den Akker1,&, Bent Brachvogel3,4, Roberto Narcisi2, Daniele Belluoccio5, Florien Jenner6, John F. Bateman5, Christopher B. Little7, Pieter Brama8, Esmeralda N. Blaney Davidson1, Peter M. van der Kraan1, Gerjo J.V.M. van Osch2,9* 1Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands 2Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands 3Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany 4Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany 5Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia 6Equine University Hospital, University of Veterinary Medicine, Vienna, Austria 7Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical Research, University of Sydney, St Leonards, New South Wales, Australia 8Veterinary Clinical Sciences, School of Veterinary Medicine, University College Dublin, Dublin, Ireland 9Department of Otorhinolaryngology, Erasmus MC University Medical Center, Rotterdam, the Netherlands &Both authors contributed equally *Corresponding author: Gerjo van Osch ([email protected]) Address for correspondence: Erasmus MC, Departments of Orthopedics and Otorhinolaryngology, Room Ee1655, Wytemaweg 80, 3015 CN Rotterdam, the Netherlands. Tel: +31-107043661. 2 Erasmus Medical Center Rotterdam █ AbsTrAcT Objective: Osteoarthritis (OA) is a joint disease characterized by progressive degen- eration of articular cartilage. Some features of OA, including chondrocyte hyper- trophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factorβ (TGFβ) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFβ-related genes are regulated during murine OA and endochondral ossification. Methods: A list of 580 TGFβ-related genes, including TGFβ signaling pathway com- ponents and TGFβ-target genes, was generated. Regulation of these TGFβ-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development). results: A total of 106 TGFβ-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplemen- tary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients. conclusion: By focusing on TGFβ-related genes during OA and chondrocyte hyper- trophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients. Keywords: osteoarthritis, TGFβ, chondrocyte hypertrophy, microarray TGFβ genes regulated during hypertrophy and OA 3 █ 1. InTrOduction Osteoarthritis (OA) is characterized by degeneration of articular cartilage and the clinical symptoms are joint pain and functional impairment [1, 2]. The chondro- cytes in articular cartilage of OA patients exhibit phenotypic changes that resemble hypertrophic differentiation of chondrocytes during endochondral ossification in the postnatal growth plate [3-8] and in embryonic joint development [9-11]. Since articular cartilage has limited repair capacity, it is essential to prevent cartilage degeneration at an early stage. To accomplish this, the pathogenic mechanisms initiating OA require further elucidation. The transforming growth factor-β (TGFβ) signaling pathway has been impli- cated in OA pathogenesis and in hypertrophic differentiation of chondrocytes [12- 15]. Polymorphisms in TGFB1 and SMAD3, a signaling molecule that is activated by binding of TGFβ to its receptor, have been associated with multiple joint patholo- gies in OA [16-20] and mutations in SMAD3 lead to early-onset of OA in multiple articular joints in humans [21, 22]. We have shown previously that protein expres- sion of Tgfb3 and phosphorylated Smad2 is reduced in two murine models for OA [23]. In mice, deficiency of Smad3, Tgfbr2 or overexpression of a truncated kinase- defective Tgfbr2, result in a degenerative joint disease resembling human OA [24, 25]. Importantly, a decrease in Tgfbr1 in murine and human articular chondrocytes correlates with OA development and elevated expression of markers for chondro- cyte hypertrophy [26]. Aside from its involvement in OA, TGFβ signaling plays a crucial role in maintenance of articular cartilage under normal physiological con- ditions and skeletal development [27, 28]. Chondrocyte-specific deletion of Tgfbr2, Tgfbr1, Smad3 or Smad4 accelerates hypertrophic differentiation of chondrocytes in the growth plate and articular cartilage [29-35]. Moreover, Tgfb2 knockout mice display severe abnormalities in bone formed by endochondral ossification [36]. Together these data indicate that TGFβ is crucial for maintenance of the articular (pre-hypertrophic) chondrocyte phenotype and that alterations in TGFβ signaling lead to chondrocyte hypertrophy and predispose to OA. It is currently not precisely known which aspects of TGFβ signaling are associ- ated with chondrocyte homeostasis, hypertrophy or OA development and whether there is overlap. A second question is whether phenotypic changes of articular chondrocytes in OA show similarity to chondrocyte hypertrophy in normal devel- opmental processes (transient growth plate cartilage, joint development). For the identification of molecular targets that may be involved in early onset and pro- gression of OA, microarray analyses have been performed on murine models for OA (early onset, trauma-induced) rather than on human OA cartilage (end-stage disease) [37-40]. One of the most recent studies in the murine OA model compared multiple independent micro-array experiments and identified the TGFβ pathway 4 Erasmus Medical Center Rotterdam as common denominator [39]. Due to the divergent effects of TGFβ signalling in articular cartilage homeostasis and disease, we hypothesized that defining the regulation of genes involved in, or regulated by, the TGFβ signaling pathway in early OA and endochondral ossification would result in the identification of novel targets implicated in phenotypic changes of chondrocytes in OA pathogenesis. In this study, we first generated a list of TGFβ-related genes, which included genes encoding components of the TGFβ signaling pathway (from the Kyoto Encyclopedia of Genes and Genomes; KEGG) [41] and genes shown to be regulated by TGFβ (i.e. TGFβ-target genes) in cartilaginous cells by microarray analyses [42-44]. Secondly, we determined which of these TGFβ-related genes are regulated in a murine model for early OA as well as during endochondral ossification in murine growth plates and/or embryonic joint development using available microarray datasets [11, 37, 45]. Finally, genes identified by this approach were further explored in the litera- ture to evaluate whether we identified genes known to be involved in murine and human OA pathology and to determine which genes might be novel candidates implicated in hypertrophic differentiation of articular chondrocytes during OA pathogenesis. █ 2. MeThOds 2.1 List of TGFβ-related genes A list of genes related to TGFβ signaling was compiled by including murine genes of the TGFβ signaling pathway derived from the KEGG database [41] and genes shown to be regulated by TGFβ in four published microarray experiments: 1x in vivo and 3x in vitro [42-44]. Takahashi et al. analyzed gene expression (Murine Genome Array U74Av2; Affymetrix) in unstimulated and TGFβ1-stimulated (10 ng/mL for 9 hours) chondrocytes from the H4 murine cell line [42, 46]. Sohn et al. performed microarray analysis (GeneChip Mouse Genome 430 2.0 Array; Affymetrix) on murine embryonic (E11.5) sclerotome cells cultured in micromass in absence or presence of TGFβ1 (5 ng/mL for 8 hours), and on vertebra isolated from wild-type and Tgbr2 knock-out mice [43]. Ramaswamy et al. evaluated gene expression (GeneChip Bovine Genome Array; Affymetrix) in micromass cultures of chondrocytes (isolated from articular cartilage of 3 cows) that were cultured in absence or presence of TGFβ1 (5 ng/mL for 8 hours) [44]. For the latter study, bovine gene probes (Affymetrix) were translated to mouse orthologs. After merging the results of the three published microarray studies [42-44], one list of 501 unique genes regulated by TGFβ (up and down)