F. Cailotto

Genome-wide association and functional studies identify the DOT1L gene to be involved in cartilage thickness and hip osteoarthritis

Hip osteoarthritis (HOA) is one of the most disabling and common joint disorders with a large genetic component that is, however, still ill-defined. To date, genome-wide association studies (GWAS) in osteoarthritis (OA) and specifically in HOA have yielded only few loci, which is partly explained by heterogeneity in the OA definition. Therefore, we here focused on radiographically measured joint-space width (JSW), a proxy for cartilage thickness and an important underlying intermediate trait for HOA. In a GWAS of 6,523 individuals on hip-JSW, we identified the G allele of rs12982744 on chromosome 19p13.3 to be associated with a 5% larger JSW (P = 4.8 × 10−10). The association was replicated in 4,442 individuals from three United Kingdom cohorts with an overall meta-analysis P value of 1.1 × 10−11. The SNP was also strongly associated with a 12% reduced risk for HOA (P = 1 × 10−4). The SNP is located in the DOT1L gene, which is an evolutionarily conserved histone methyltransferase, recently identified as a potentially dedicated enzyme for Wnt target-gene activation in leukemia. Immunohistochemical staining of the DOT1L protein in mouse limbs supports a role for DOT1L in chondrogenic differentiation and adult articular cartilage. DOT1L is also expressed in OA articular chondrocytes. Silencing of Dot1l inhibited chondrogenesis in vitro. Dot1l knockdown reduces proteoglycan and collagen content, and mineralization during chondrogenesis. In the ATDC5 chondrogenesis model system, DOT1L interacts with TCF and Wnt signaling. These data are a further step to better understand the role of Wnt-signaling during chondrogenesis and cartilage homeostasis. DOT1L may represent a therapeutic target for OA.

Tight regulation of wingless-type signaling in the articular cartilage - subchondral bone biomechanical unit: transcriptomics in Frzb-knockout mice

IntroductionThe aim of this research was to study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled-related protein (Frzb) compared to wild-type mice by transcriptome analysis.MethodsGene-expression analysis of the articular cartilage and subchondral bone of three wild-type and three Frzb-/- mice was performed by microarray. Data from three wild-type and two Frzb-/- samples could be used for pathway analysis of differentially expressed genes and were explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the wingless-type (WNT) pathway was analysed using Western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micro-masses and mouse ribcage chondrocytes.ResultsExtracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were up-regulated in Frzb-/- samples. Several WNT receptors, target genes and other antagonists were up-regulated, but no difference in active β-catenin was found. Analysis of ATDC5 cell micro-masses overexpressing FRZB indicated an up-regulation of aggrecan and Col2a1, and down-regulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in down-regulation of aggrecan and Col2a1. Pathways associated with cell cycle were down-regulated in this transcriptome analysis. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells.ConclusionsOur analysis provides evidence for tight regulation of WNT signalling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint.

Genome-wide association and functional studies identify a role for IGFBP3 in hip osteoarthritis

Objectives To identify genetic associations with hip osteoarthritis (HOA), we performed a meta-analysis of genome-wide association studies (GWAS) of HOA. Methods The GWAS meta-analysis included approximately 2.5 million imputed HapMap single nucleotide polymorphisms (SNPs). HOA cases and controls defined radiographically and by total hip replacement were selected from the Osteoporotic Fractures in Men (MrOS) Study and the Study of Osteoporotic Fractures (SOF) (654 cases and 4697 controls, combined). Replication of genome-wide significant SNP associations (p ≤5×10−8) was examined in five studies (3243 cases and 6891 controls, combined). Functional studies were performed using in vitro models of chondrogenesis and osteogenesis. Results The A allele of rs788748, located 65 kb upstream of the IGFBP3 gene, was associated with lower HOA odds at the genome-wide significance level in the discovery stage (OR 0.71, p=2×10−8). The association replicated in five studies (OR 0.92, p=0.020), but the joint analysis of discovery and replication results was not genome-wide significant (p=1×10−6). In separate study populations, the rs788748 A allele was also associated with lower circulating IGFBP3 protein levels (p=4×10−13), suggesting that this SNP or a variant in linkage disequilibrium could be an IGFBP3 regulatory variant. Results from functional studies were consistent with association results. Chondrocyte hypertrophy, a deleterious event in OA pathogenesis, was largely prevented upon IGFBP3 knockdown in chondrocytes. Furthermore, IGFBP3 overexpression induced cartilage catabolism and osteogenic differentiation. Conclusions Results from GWAS and functional studies provided suggestive links between IGFBP3 and HOA.

DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

Osteoarthritis is the most prevalent and crippling joint disease, and lacks curative treatment, as the underlying molecular basis is unclear. Here, we show that DOT1L, an enzyme involved in histone methylation, is a master protector of cartilage health. Loss of DOT1L disrupts the molecular signature of healthy chondrocytes in vitro and causes osteoarthritis in mice. Mechanistically, the protective function of DOT1L is attributable to inhibition of Wnt signalling, a pathway that when hyper-activated can lead to joint disease. Unexpectedly, DOT1L suppresses Wnt signalling by inhibiting the activity of sirtuin-1 (SIRT1), an important regulator of gene transcription. Inhibition of SIRT1 protects against osteoarthritis triggered by loss of DOT1L activity. Modulating the DOT1L network might therefore be a therapeutic approach to protect the cartilage against osteoarthritis.

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis

BackgroundEffective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.MethodsLung fibrosis was induced in wild-type or noggin haploinsufficient (Nog+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.ResultsNog+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.ConclusionsUpon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.

Osteogenesis induced by frizzled-related protein (FRZB) is linked to the netrin-like domain

Abnormal Wnt signaling is associated with bone mass disorders. Frizzled-related protein (FRZB, also known as secreted frizzled-related protein-3 (SFRP3)) is a Wnt modulator that contains an amino-terminal cysteine-rich domain (CRD) and a carboxy-terminal Netrin-like (NTN) motif. Frzb−/− mice show increased cortical thickness. However, the direct effect of FRZB on osteogenic differentiation and the involvement of the structural domains herein are not fully understood. In this study, we observed that stable overexpression of Frzb in MC3T3-E1 cells increased calcium deposition and osteoblast markers compared with control. Western blot analysis showed that the increased osteogenesis was associated with reduced canonical, but increased non-canonical Wnt signaling. On the contrary, loss of Frzb induced the opposite effects on osteogenesis and Wnt signaling. To translationally validate the positive effects of FRZB on primary human cells, we treated human periosteal and human bone marrow stromal cells with conditioned medium from MC3T3-E1 cells overexpressing Frzb and observed an increase in Alizarin red staining. We further studied the effect of the domains. FrzbNTN overexpression induced similar effects on osteogenesis as full-length Frzb, whereas FrzbCRD overexpressing cells mimicked loss of Frzb experiments. The CRD is considered as the Wnt binding domain, but the NTN domain also has important effects on bone biology. FRZB and other SFRPs or their specific domains may hold surprising potential as therapeutics for bone and joint disorders considering that excess of SFRPs has effects that are not expected under physiological, endogenous expression conditions.

A5.6 Cholecystokinin and purinoreceptor antagonists modulate OA-associated GPR22 signalling

Background Osteoarthritis (OA) is the most common type of arthritis and its prevalence increases in the ageing population. A genome-wide association study (GWAS) identified an OA susceptibility locus of approximately 500 kb on chromosome 7q22. This locus contains a linkage disequilibrium block of six genes that includes G protein coupled receptor protein 22 (GPR22). GPR22 signalling inhibits protein kinase A (PKA) activity but its ligand is unknown. GPR22 shares 37% sequence homology with the Cholecystokinin B receptor (CCK-BR) and is predicted to belong to the purinergic P2Y12 family. Here we aim to further study the role of GPR22 in cartilage. Methods We used the ATDC5 chondrogenic cell line as model system. Stably overexpressing GPR22 cells (GPR22+) were cultured in triplicate as high density micromasses (200 000 cells/10 µl). Cells were treated daily with ITS in DMEM/F12 medium for 14 days to induce chondrogenesis. Mineralization was triggered with beta-glycerol-phosphate in α-MEM medium till day 21. Different chemical compounds were added: CCK-BR antagonist AG-041R (1 and 10 µM), broad-spectrum purinoreceptor antagonist suramin (10, 33 and 100 µM) and PKA activator 8-Br-cAMP (10 and 33 µM). Gene expression of CCK-BR and chondrogenic markers, including Aggrecan (Agg), Decorin (Dco), Versican (Vcan), Collagen type 2 (Col2a1) and type 10 (Col10a1) and Matrix metalloprotease 13 (MMP13), were analysed. Sulfated proteoglycan, mineralization and collagen content were quantified by Alcian Blue, Alizarin Red, Safranin O and Picosirius Red staining. Result Overexpression of GPR22 stimulates chondrocyte hypertrophy and accelerates calcification. CCK-BR expression was not detected in ATDC5 cells. AG-041R 1 µM increased Agg, Col2a1, Col10a1 expression at day 1 compared to unchallenged cells. Additionally, on day 21 the amount of proteoglycan and calcium deposition was increased. The use of 10 µM AG-041R and of 100 µM suramin was toxic for cells. However, 10 µM suramin treatment lead to increased decorin and versican expression. This translated into increased proteoglycan (and collagen) levels in the extracellular matrix (ECM). 8-Br-cAMP increased mRNA expression of Agg, Col2a1 and Col10a1. Conclusion A cholecystokinin antagonist and the broad spectrum purinoreceptor antagonist slightly compensate for the effect of GPR22 overexpression on chondrogenesis. The PKA activator, 8-Br-cAMP did not change the GPR22 phenotype at the ECM level, but induced subtle transcriptomic changes.

A5.12 SFRPS in cartilage biology: more than just WNT antagonists

Background and Objectives Wnts play an important role in cartilage development, homeostasis and degeneration. Therefore, exploring the therapeutic potential of factors that can modulate Wnts is of great interest. Secreted frizzled related proteins (SFRPs) were identified as secreted Wnt antagonists. Polymorphisms in Frizzled-related protein (FRZB-SFRP3) are associated with osteoarthritis (OA). Here, we study the role of SFRPs and their structural domains in cartilage biology. Materials and Methods Surgical destabilization of the medial meniscus (DMM) was performed on eight-week-old male Frzb -/-, Sfrp1-/- and wild-type mice. Eight weeks after surgery, mice were sacrificed and histological scores were determined for the femoral and tibial articular surfaces following the OARSI guidelines. Chondrogenesis of ATDC5 micromasses, stably overexpressing pcDNA3.1-FRZB, -FRZB-ΔCRD, -FRZB-ΔNTN, -SFRP1, -SFRP1-ΔCRD, -SFRP1-ΔNTN, pGIPZ-shmiRNA-Frzb or control plasmids, was induced by culturing cells for 14 days in DMEM/F12 + 5%FBS, supplemented with ITS (insulin, transferrin, sodium selenite). On day 14, cells were switched to mineralization medium (αMEM + 5%FBS, supplemented with ITS, β-glycerophosphate and ascorbic acid). mRNA expression of chondrogenic markers (Aggrecan, Col2a1 and Col10a1) were assessed by quantitative RT-PCR. Western blot was used to study canonical (β-catenin) and non-canonical (CamKII) Wnt signalling. Results The OARSI score showed a significant increase in cartilage damage in DMM-operated Frzb -/-mice compared to wild-type but not in Sfrp1-/- mice. Overexpression of Frzb in ATDC5 micromasses boosted chondrogenesis with up-regulation of Col2a1, whereas Frzb silencing lead to decreased chondrogenesis. These results corresponded with a reduction or increase in the activation of canonical WNT signalling, respectively. Wnt/CamKII signalling was not affected. SFRP1 overexpression led to a decrease in Col2a1 transcription, but to an increase in Col10a1 expression. These results corresponded with increased activation of canonical WNT signalling pathway and decreased phosphorylation of CamKII. Domain studies for Frzb and Sfrp1 showed that overexpression of the NTN domain increased expression of Col2a1 and Aggrecan. Conclusions Different SFRPs have a strong, but distinct effect on cartilage. Frzb promoted early chondrogenesis through modulation of canonical Wnt signalling. Sfrp1 reduced early chondrogenesis and favoured hypertrophy by affecting both canonical and non-canonical Wnt signalling. Domain studies revealed that the NTN domain of SFRPs is responsible for the positive effect on chondrogenesis.

A5.5 SMOC2 modulates chondrogenesis by interfering with WNT and BMP signalling

Background and Objectives A proteomic analysis of cartilage revealed an increase in SPARC-related modular calcium binding protein-2 (SMOC2) in patients with osteoarthritis (OA). SMOC2 was originally isolated from a chondrogenic extract of articular cartilage together with GDF5 and FRZB, proteins associated with OA. We investigated SMOC2 in chondrocyte differentiation. Methods Three independent stable clonal colonies of the ATDC5 chondrogenic cell line, control (empty vector/GIPZ) and SMOC2 overexpressing (SMOC2+) or knocked-down cells (SMOC2-), were used. Clones were cultured as high density micromasses (2x105 cells/10µl). Chondrogenesis was induced by culturing cells for 14 days (D) in DMEM/F12 + 5%FBS + ITS [insulin (10µg/ml), transferrin (10µg/ml) and sodium selenite (30nM)]. On D14, cells were switched to mineralization medium (αMEM+5%FBS+ITS). mRNA expression of markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens was assessed by quantitative RT-PCR. Likely, we assessed mRNA expression of Wnts (-3a, -4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralised content respectively. Western blot was used to study BMP and Wnts pathways. Results In SMOC2 + cells, during the early (D1-7) and late proliferation phase (D7-14), Col2a1 and Agg mRNA increased less than controls. Safranin O and Alcian blue staining were also less upregulated in SMOC2 + . This was consistent with lower activation of Smad 1/5/8 in SMOC2 + . Col10a1 mRNA increased as expected from D14 onwards, but significantly less in SMOC2 + compared to controls. SMOC2 overexpression affected Wnt mRNA levels. During mineralization (D14-21), Col10a1 mRNA was strongly increased, but to a lesser extent in SMOC2 + , and alizarin red was much stronger in controls. The SMOC2- cells exhibited opposite features as SMOC2 + . In the D1-D14 phases, Collagen and Proteoglycan content was higher than in controls, alike Col2a1 mRNA level. In the D14-21 phase, these differences were maintained, and Col10a1 mRNA level was higher in SMOC2-, and a trend was found for a higher mineralization content in SMOC2-. No difference was detected in cell viability for both SMOC2 + and SMOC2- compared to their respective controls. Conclusions SMOC2 modulates chondrogenesis by affecting BMP-Smad and Wnt signalling. These data suggest that SMOC2 can play a modulating role in OA.

SMOC2 inhibits calcification of osteoprogenitor and endothelial cells

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2’s inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders.