Genetic association study of UCMA/GRP and OPTN genes (PDB6 locus) with Paget's disease of bone

We performed a genetic association study of rare variants and single nucleotide polymorphisms (SNPs) of UCMA/GRP and OPTN genes, in French-Canadian patients with Paget's disease of bone (PDB) and in healthy controls from the same population. We reproduced the variant found in the UCMA/GRP basal promoter and tested its functionality using in vitro transient transfection assays. Interestingly, this SNP rs17152980 appears to affect the transcription level of UCMA/GRP. In addition, we have identified five rare genetic variants in UCMA/GRP gene, four of them being population-specific, although none were found to be associated with PDB. Six Tag SNPs of UCMA/GRP gene were associated with PDB, particularly the SNP rs17152980 (uncorrected P = 3.8 x 10(-3)), although not significant after Bonferroni's correction. More importantly, we replicated the strong and statistically significant genetic association of two SNPs of the OPTN gene, the rs1561570 (uncorrected P = 5.7 x 10(-7)) and the rs2095388 (uncorrected P = 4.9 x 10(-3)), With PDB. In addition, we identified a very rare variant found to be located close to the basal promoter of the OPTN gene, at -232 bp from its distal transcription start site. Furthermore, depending on the type of allele present (G or A), the binding of several important nuclear factors such as the vitamin D or the retinoic acid receptors is predicted to be altered at this position, suggesting a significant effect in the regulation of transcription of the OPTN gene. In conclusion, we identified a functional SNP located in the basal promoter of the UCMA/GRP gene which provided a weak genetic association with PDB. In addition, we replicated the strong genetic association of two already known SNPs of the OPTN gene, with PDB in a founder effect population. We also identified a very rare variant in the promoter of OPTN, and through bioinformatic analysis, identified putative transcription factor binding sites likely to affect OPTN gene transcription. (C) 2012 Elsevier Inc. All rights reserved.Fonds de la Recherche du Quebec - Sante (FRQS), Canada; Portuguese Science and Technology Foundation, Portugal [SFRH/BPD/48206/2008]; Catalyst Grant (Bone Health) from the Canadian Institutes of Health Research (Canada); CHUQ Foundation (Canada); Groupe de Recherche en Maladies Osseuses (Canada); Canadian Foundation for Innovation (Canada); FRSQ (Canada); Laval University (Canada); CHUQ (CHUL) Research Centre (Canada); Centre of Marine Sciences (CCMAR) (Portugal)info:eu-repo/semantics/publishedVersio

Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes

To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2CCN2 and CCN2CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 x 10-9 m for CCN2 and CCN2, and 1.95 x 10-9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent proteinCCN2 and HaloCCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.NWO ZonMw [MKMD 40-42600-98-13007]; FCT [SFRH/BPD/70277/2010]info:eu-repo/semantics/publishedVersio

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: implications for calcification-related chronic inflammatory diseases

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and gamma-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein gamma-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNF alpha, IL-1 beta and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.Portuguese Science and Technology Foundation (FCT) [PTDC/SAU-ORG/117266/2010, PTDC/BIM-MEC/1168/2012, UID/Multi/ 04326/2013]; FCT fellowships [SFRH/BPD/70277/2010, SFRH/BD/111824/2015

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1

Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1 WD) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1 WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1 WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1 WD at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG-and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients. © 2011 Nature America, Inc. All rights reserved

Structure, expression and function of the novel cartilage specific protein Ucma

Die Umwandlung von Knorpel in Knochengewebe bei der Skelettentwicklung – die enchondrale Ossifizierung – ist ein komplexer Vorgang, dessen Verständnis auch für pathologische Vorgänge, wie Wachstumsstörungen, Skelettdysplasien und Osteoarthrose von Bedeutung ist. Bei diesem Vorgang spielt insbesondere die Differenzierung von Knorpelzellen – Chondrozyten – eine wichtige Rolle. Die Stadien der Chondrozytenreifung können durch Gene charakterisiert werden, die differentiell in den einzelnen Reifungszonen wie Ruhezone, Proliferationszone und hypertopher Zone exprimiert sind. Der Schwerpunkt dieser Arbeit versteht sich in der Analyse von zwei differentiell exprimierten Genen, TSG und Ucma in der Knorpelentwicklung. Im ersten Teil dieser Arbeit werden Versuche zur Aufklärung der Expression und Funktion von Twisted Gastrulation (TSG) einem bekannten Regulator des BMP (bone morphogenetic protein) Signaltransduktionswegs beschrieben. TSG wird während der Chondrozytendifferenzierung vermehrt in hypertrophen Chondrozyten, aber auch in der Ruhezone exprimiert (54). In in vitro Experimenten mit der chondrogenen MC615 Zelllinie (43) und isolierten primären Rippenchondrozyten fungierte TSG als BMP Antagonist hinsichtlich der Induktion der Expression von Kollagen II (Col2a1) und Kollagen X (Col10a1) RNA sowie der Phosphorylierung der BMP-abhängigen Smads (1,5,8). Die knorpelspezifische Überexpression von TSG unter dem Col2a1 Promotor zeigte eine reduzierte Col10a1 Expression, aber keine morphologischen Auffälligkeiten. Im Rahmen der in vitro Experimente mit TSG wurde die Notwendigkeit von stabilen und besser charakterisierten Zelllinien zur Analyse der Chondrozytendifferenzierung deutlich. Deshalb wurde die MC615 Zelllinie subkloniert, und die Subklone wurden hinsichtlich ihres Expressionsprofils sowie ihrer BMP Responsivität charakterisiert. Desweiteren wurden bei ausgesuchten Klonen das endogene Differenzierungspotential sowie die bei Differenzierung durch Langzeitkultur auftretenden Veränderungen in Matrixzusammensetzung und Expression von Markergenen analysiert. Dadurch konnten Zelllinien mit verschiedenen Differenzierungszuständen und unterschiedlichem Differenzierungspotential etabliert werden. Der Großteil dieser Arbeit widmet sich der Untersuchung eines bisher nicht bekannten differentiell epxrimierten Gens, das von Uwe Dietz im Rahmen einer Studie über Retinsäure induzierte Dedifferenzierung von murinen Chondrozyten gefunden wurde. Dieses Gen, im weiteren Ucma (unique cartilage matrix associated protein bzw. nach den Entdeckern Uwe; Cordula, Michael und Andreas) genannt, codiert für ein kurzes sezerniertes Protein, welches hauptsächlich in der Matrix von distalen Chondrozyten der Ruhezone vorkommt. Es ist in Wirbeltieren hochkonserviert, allerdings konnte im Huhngenom kein Ortholog identifiziert werden. Das rekombinant epxrimierte Protein wird vermutlich durch eine Subtilisin ähnliche Proproteinkonvertase weiter prozessiert und liegt dann in einer 9,5kDa großen tyrosinsulfatierten Form vor. Ucma wird ab Embryonaltag 13,5 hauptsächlich in Ruhezonenchondrozyten der Wirbelsäule und wenig später auch in Rippen- und Epiphysenknorpel exprimiert. Im Laufe der Embryonalentwicklung nimmt die Expressionsstärke vom Ucma zunächst zu; postnatal schwächt sich die Expression jedoch wieder deutlich ab. Auch in Zelllinien und den etablierten Differenzierungsmodellen von MC615 Subklonen ist die Expression von Ucma auf differenzierte Chondroyzten beschränkt, die aber noch nicht hypertrophiert sind. Dieses reifungsabhängige Abschalten der Expression könnte durch die Repression der Ucma Expression durch BMP-2, die in vitro nachgewiesen werden konnte, reguliert werden. Um die Rolle von Ucma in vivo zu studieren wurde es knorpelspezifisch unter dem Col2a1 Promotor überexprimiert. Die Analyse von 12 transgenen Mauslinien ergab keine auf die Expression des Transgens zurückzuführenden morphologischen Veränderungen. Allerdings war die Überexpression generell eher schwach ausgeprägt und daher das Ausbleiben eines offensichtlichen Phänotyps nicht verwunderlich. Zur Analyse der Funktion von Ucma in vitro wurde der Effekt des rekombinanten Proteins in verschiedenen Zellkultursystemen getestet. Dabei konnte keine Wirkung von Ucma auf Chondrozytendifferenzierung, Expressionsprofil und Zellproliferation beobachtet werden. Interessanterweise inhibierte Ucma die Differenzierung von MC3T3 Präosteoblasten zu Osteoblasten, was auf eine mögliche Rolle von Ucma als Vermittler zwischen Knorpel- und Knochendifferenzierung hindeutet.The replacement of cartilage by bone during skeletal development is a complex process called endochondral ossification. Elucidation of mechanisms underlying endochondral ossification and in particular chondrocyte differentiation could help to understand pathologenic mechanisms of growth retardation, in skeletal dysplasias and of osteoarthritis. The steps of chondrocyte differentiation are characterised by differentially expressed genes in resting, proliferating and hypertrophic chondrocytes. The aim of this work was to elucidate the role of two differentially expressed genes in cartilage development, TSG and Ucma. In the first part I report on the expression and function of twisted gastrulation (TSG), a known BMP (bone morphogenetic protein) regulator. During chondrocyte differentiation TSG was most abundant in hypertrophic chondrocytes; but it was also found in the resting zone. Recombinant TSG impaired BMP dependent Smad phosphorylation and subsequent Induction of Col2a1 and Col10a1 expression in MC615 cells and primary murine rib chondrocytes in vitro. Cartilage specific overexpression of TSG in mice under the Col2a1 promoter led to decreased Col10a1 expression but did not result in altered growth plate morphology. During the experiments with the MC615 cell line the need for stable and better characterised murine chondrocyte cell lines became apparent. Thus the MC615 cell line was subcloned and expression profiles as well as BMP-2 responsivity of the emerging subclones were determined. The differentiation capacity of selected clones was assessed by monitoring matrix formation and expression of marker genes in long term culture. Thereby chondrocyte cell lines of distinct differentiation status and capacity could be established. The main part of this work deals with the characterisation of a so far unknown differentially expressed gene, called Ucma. This gene was initially found by Uwe Dietz to be repressed during retinoic acid induced dedifferentiation of primary murine chondrocytes. The gene product was termed Ucma - unique cartilage matrix associated protein – for the main site of its occurrence. The Ucma gene is highly conserved among vertebrates exept chicken and codes for a short secreted protein localised in the matrix of distal chondrocytes. Recombinantly expressed Ucma was cleaved at a predicted subtilisin like proprotein convertase (SPC) recognition site. The resulting 9,5kDa protein is hydrophilic and was shown to be sulphated at distinct tyrosine residues. First Ucma mRNA expression was detected in vertebrae of E13,5 mouse embryos. Its maximum expression was observed in distal epiphyseal and vertebral chondrocytes between E18,5 and P2, declining later on. During differentiation of an MC615 subclone in vitro Ucma paralleled largely the expression of collagen II, and decreased with maturation to hypertrophic cells; it was downregulated by BMP-2. Thus it marks an early, distinct differentiation stage of chondrocytes. Recombinant Ucma did not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interfered with osteogenic differentiation of MC3T3 preosteoblasts. Transgenic expression of Ucma in cartilage under the Col2a1 promoter did not show an obvious phenotype, most probable due to insufficient overexpression. Thus Ucma defines a new population of juvenile chondrocytes and may play a role in the assembly of cartilage as well as in the regulation of endochondral ossification