Daniele Belluoccio

Mutations in TRPV4 cause an inherited arthropathy of hands and feet

Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.

S100A8 and S100A9 in experimental osteoarthritis

IntroductionThe objective was to evaluate the changes in S100A8 S100A9, and their complex (S100A8/S100A9) in cartilage during the onset of osteoarthritis (OA) as opposed to inflammatory arthritis.MethodsS100A8 and S100A9 protein localization were determined in antigen-induced inflammatory arthritis in mice, mouse femoral head cartilage explants stimulated with interleukin-1 (IL-1), and in surgically-induced OA in mice. Microarray expression profiling of all S100 proteins in cartilage was evaluated at different times after initiation of degradation in femoral head explant cultures stimulated with IL-1 and surgically-induced OA. The effect of S100A8, S100A9 or the complex on the expression of aggrecan (Acan), collagen II (Col2a1), disintegrin and metalloproteases with thrombospondin motifs (Adamts1, Adamts 4 &Adamts 5), matrix metalloproteases (Mmp1, Mmp3, Mmp13 &Mmp14) and tissue inhibitors of metalloproteinases (Timp1, Timp2 &Timp3), by primary adult ovine articular chondrocytes was determined using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsStimulation with IL-1 increased chondrocyte S100a8 and S100a9 mRNA and protein levels. There was increased chondrocyte mRNA expression of S100a8 and S100a9 in early but not late mouse OA. However, loss of the S100A8 staining in chondrocytes occurred as mouse OA progressed, in contrast to the positive reactivity for both S100A8 and S100A9 in chondrocytes in inflammatory arthritis in mice. Homodimeric S100A8 and S100A9, but not the heterodimeric complex, significantly upregulated chondrocyte Adamts1, Adamts4 and Adamts 5, Mmp1, Mmp3 and Mmp13 gene expression, while collagen II and aggrecan mRNAs were significantly decreased.ConclusionsChondrocyte derived S100A8 and S100A9 may have a sustained role in cartilage degradation in inflammatory arthritis. In contrast, while these proteins may have a role in initiating early cartilage degradation in OA by upregulating MMPs and aggrecanases, their reduced expression in late stages of OA suggests they do not have an ongoing role in cartilage degradation in this non-inflammatory arthropathy.

Global comparative transcriptome analysis of cartilage formation in vivo.

BackgroundDuring vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of in vivo gene expression.ResultsTibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for Agc1, Bmp8a, Col2a1, Fgfr4, Foxa3, Gdf5, Klf2, Klf4, Lepre1, Ncad, Sox11, and Trpv4. Further, independent validation of the microarray data was achieved by in situ hybridization to analyse the expression of Lepre1, Pcdh8, Sox11, and Trpv4 from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several Bmp, Gdf, Wnt, Sox and Fox family members.ConclusionThese data represent the first global gene expression profiling analysis of chondrogenic tissues during in vivo development. They identify genes for further study on their functional roles in chondrogenesis, and provide a comprehensive and important resource for future studies on cartilage development and disease.

Proteomic characterization of mouse cartilage degradation in vitro.

OBJECTIVE To develop proteomics to analyze mouse cartilage degradation and correlate transcriptional and translational responses to catabolic stimuli. METHODS Proteomic techniques were used to analyze catabolism in mouse femoral head cartilage. Using specific methods to prepare cartilage extracts and conditioned media for 2-dimensional polyacrylamide gel electrophoresis and subsequent tandem mass spectrometry, we identified novel proteins and fragments released into the media of control, interleukin-1alpha (IL-1alpha)-treated, and all-trans-retinoic acid (RetA)-treated explants. Fluorescence 2-dimensional difference gel electrophoresis was used to quantify protein expression changes. We also measured changes in messenger RNA (mRNA) expression to distinguish transcriptional and posttranslational regulation of released proteins. RESULTS Differentially abundant proteins in the media of control and treated explants included fragments of thrombospondin 1 and connective tissue growth factor. IL-1alpha stimulated release of the cartilage degeneration marker matrix metalloproteinase 3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of the extracellular matrix proteins cartilage oligomeric matrix protein, link protein, and matrilin-3 into the media, which was accompanied by a dramatic reduction in the corresponding mRNA transcript levels. Gelsolin, which has been implicated in cytoskeletal reorganization in arthritis synovial fibroblasts but has not been previously associated with cartilage pathology, was regulated by IL-1alpha and RetA. CONCLUSION In this first analysis of mouse cartilage degradation and protein release using proteomics, we identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and genetically modified mouse cartilage will provide insights into the mechanisms of cartilage degeneration.

Proteomic analysis of cartilage proteins

While the analysis of the cartilage proteome is important for our comprehensive understanding of the development and disease of this important tissue, several unique features of cartilage present some technical obstacles. Firstly, cartilage is difficult to obtain in adequate quantities for many protein analyses, especially from mice which are otherwise powerful experimental models. Furthermore, the cartilage extracellular matrix contains an insoluble network of collagen II-containing fibrils that are integrated within an abundant anionic network of aggrecan and hyaluronan aggregates. These interacting networks provide a structural scaffold for the covalent and non-covalent attachment of other proteins and glycoproteins. Consequently, proteomic analysis of cartilage requires extraction of proteins with chaotropic agents to achieve and significant protein solubilization. Finally, isolated chondrocytes are phenotypically unstable, which requires rapid isolation of cells or the use of specific culture conditions. Despite these problems, recent improvements in the sensitivity and reproducibility of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) techniques, combined with improved tissue preparation and sample pre-fractionation approaches, have made the proteomic characterization of cartilage tissues possible. Here we review the approaches that have been used and describe in detail protocols for the proteomic analysis of cartilage tissues and cells.

Cartilage intermediate layer protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis.

Using transcriptome profiling to determine differential gene expression between the permanent mouse articular cartilage and the transient growth plate cartilage, we identified a highly expressed gene, Cilp2, which is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases. We demonstrated that Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix. mRNA expression analysis indicated that whereas Cilp1 and Cilp2 are expressed most abundantly in cartilaginous tissues, expression can be detected in muscle and heart.

Transcriptomics of Wild‐Type Mice and Mice Lacking ADAMTS‐5 Activity Identifies Genes Involved in Osteoarthritis Initiation and Cartilage Destruction

OBJECTIVE To identify changes in gene expression in mice with osteoarthritis (OA) in order to explore the mechanisms of the disease. METHODS Gene expression profiling was performed in cartilage from mice with surgically induced OA. We used wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is lacking and aggrecan loss and cartilage erosion are inhibited, to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown. Mechanical instability was introduced into the knee joints of 10-week-old male mice via surgical destabilization of the medial meniscus (DMM). Cartilage from the developing lesion in the destabilized medial meniscus and corresponding regions in sham-operated joints was harvested by microdissection at 1, 2, and 6 weeks postsurgery, and RNA was extracted, amplified, and hybridized to whole-genome microarrays. RESULTS Several previously identified OA-related genes, including Ptgs2, Crlf1, and Inhba, and novel genes, such as Phdla2 and Il11, were up-regulated in both WT mice and Adamts5Δcat mice, indicating that they are independent of ADAMTS-5 activity. The altered expression of other genes, including Col10a1, the sentinel marker of cartilage hypertrophy, and Wnt/β-catenin pathway genes, required ADAMTS-5 activity. Cell death pathway genes were dysregulated, and Tp53, Foxo4, and Xbp1 endoplasmic reticulum-stress transcriptional networks were activated. Analysis of degradome genes identified up-regulation of many proteases, including Mmp3, Capn2, and the novel cartilage proteases Prss46 and Klk8. Comparison with other studies identified 16 genes also dysregulated in rat and human OA as priorities for study. CONCLUSION We have identified, for the first time, several genes that have an ADAMTS-5-independent role in OA, identifying them as possible OA initiation candidates. This work provides new insights into the sequence of gene dysregulation and the molecular basis of cartilage destruction in OA.

A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage.

In vertebrates, longitudinal bone growth is the consequence of a complex series of events that take place in a specialized structure, the growth plate cartilage. Within the growth plate chondrocytes undergo a sequential maturation program from resting cells to proliferative, pre-hypertrophic, and ultimately hypertrophic end-stage chondrocytes. This process of chondrocyte maturation is under the control of the temporally and spatially regulated expression of a myriad of signaling molecules, transmembrane receptors, transcription factors, and structural extracellular matrix (ECM) proteins. One approach to the comprehensive definition of the key components of such complex interrelated pathways is the use of microarray expression profiling to catalogue transcriptome changes during chondrocyte maturation in the individual developmental zones of the mouse growth plate cartilage. However, this has not been achieved because of the difficulty in obtaining sufficient quantities of the individual growth plate cartilage zones to all microarray analysis. In this study we describe the development of microdissection methods for the isolation of tissue from the proliferative, pre-hypertrophic, and proliferative zone from one single mouse femur, RNA extraction and linear amplification of the RNA to allow interrogation of NIA 15k microarrays to generate comparative expression profiles. Verification of a subset of differentially expressed genes by RT-PCR and by in situ hybridization confirmed the reliability of this approach.

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status.

Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone‐specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate.