Molecular basis for differences between human joints

The molecular program of a cell determines responses including induction or inhibition of genes for function and activity, and this is true of the cells within articular cartilage, a major functional component of the joint. While our studies have previously focussed on differences in the molecular programs of the cells within the superficial and deep zones, we have recently begun to focus on relative differences between joints, such as the knee and ankle. In the human, these joints vary greatly in their susceptibility to joint diseases, such as osteoarthritis (OA). We have predicted that there would be a molecular basis for differences between joints that could lead to differences in susceptibility to OA, if inherent pathways locked into the resident cells induce differences in their response to their environment. We have been able to show that there are differences between the matrix components and water content; these properties correspond to a higher equilibrium modulus and dynamic stiffness but lower hydraulic permeability and serve to make the ankle cartilage stiffer, slowing movement of molecules through the cartilage. In addition to these biochemical differences in the cartilage matrix, we have also identified relative differences in the strength of the response to stimulation of chondrocytes from knee and ankle. The stronger response of the knee chondrocytes includes factors that increase damage to the cartilage matrix, such as a depression of matrix synthesis and increased enzyme activity. This response by the knee chondrocytes results in enzyme damage to the matrix that the cells may not be able to repair, while the weaker response of the ankle chondrocytes may allow the cells to repair their matrix damage.     

Noncollagenous, nonproteoglycan macromolecules of cartilage

Extracellular matrix comprises approximately 90% of cartilage, with collagens and proteoglycans making up the bulk of the tissue. In recent years, several abundant cartilage proteins that are neither collagens nor proteoglycans have been characterized in detail. The putative roles of these proteins range from involvement in matrix organization or matrix-cell signaling (PRELP, chondroadherin, cartilage oligomeric protein and cartilage matrix protein) through to molecules that are likely to be involved with modulation of the chondrocyte phenotype (CD-RAP, CDMPs, chondromodulin and pleiotrophin). Other molecules, such as the cartilage-derived C-type lectin and cartilage intermediate layer protein have no role as yet. Due to the difficulties associated with experimentally manipulating a tissue that is 90% extracellular matrix in a manner that can be readily transferred to the whole organism, many of these molecules have been focused on by a surprisingly small number of researchers. This review focuses on newly discovered proteins and glycoproteins in cartilage, with a bias towards those that have structural roles or that are unique to cartilage. Received 7 January 1999; accepted 11 March 1999     

Molecular pathology and pathobiology of osteoarthritic cartilage

The biochemical properties of articular cartilage rely on the biochemical composition and integrity of its extracellular matrix. This matrix consists mainly of a collagen network and the proteoglycan-rich ground substance. In osteoarthritis, ongoing cartilage matrix destruction takes place, leading to a progressive loss in joint function. Beside the degradation of molecular matrix components, destabilization of supramolecular structures such as the collagen network and changes in the expression profile of matrix molecules also take place. These processes, as well as the pattern of cellular reaction, explain the pathology of osteoarthritic cartilage degeneration. The loss of histochemical proteoglycan staining reflects the damage at the molecular level, whereas the supramolecular matrix destruction leads to fissuring and finally to the loss of the cartilage. Chondrocytes react by increasing matrix synthesis, proliferating, and changing their cellular phenotype. Gene expression mapping in situ and gene expression profiling allows characterization of the osteoarthritic cellular phenotype, a key determinant for understanding and manipulating the osteoarthritic disease process.     

Cartilage biology, pathology, and repair

Osteoarthritis is one of the most common forms of musculoskeletal disease and the most prominent type of arthritis encountered in all countries. Although great efforts have been made to investigate cartilage biology and osteoarthritis pathology, the treatment has lagged behind that of other arthritides, as there is a lack of effective disease-modifying therapies. Numerous approaches for dealing with cartilage degradation have been tried, but enjoyed very little success to develop approved OA treatments with not only symptomatic improvement but also structure-modifying effect. In this review we discuss the most recent findings regarding the regulation of cartilage biology and pathology and highlight their potential therapeutic values.     

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.     

Structural studies of a phosphatidyl serine-amorphous calcium phosphate complex

A phosphatidyl serine-amorphous calcium phosphate complex has been synthesized as a model of the matrix vesicle system that is associated with the induction of mineral deposition in bone, cartilage and dentine. The complex has been studied using a novel technique of subtractive extended X-ray absorption fine structure (EXAFS). This enables spectra of the components of the molecules to be subtracted from the complex so as to identify the sites of interaction. The results suggest there is a movement in the nitrogen atom of the phosphatidyl serine which approaches the calcium atom in the mineral phase. This interpretation would link the membrane structure of the vesicle to the structure of the mineral in a way that could explain some of its roles in biomineralization. Received 14 November 1997; accepted 23 December 1997     

In vivo matrix-guided human mesenchymal stem cells

Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation. Received 3 November 2005; received after revision 15 December 2005; accepted 4 January 2006     

Cytokines and proteoglycans

Cytokines play an important regulatory role in the metabolism of proteoglycans. Proteoglycans are found in plasma membranes, but predominantly in the extra-cellular matrix. In the latter they are quantitatively and qualitatively essential components. Especially in a tissue like cartilage without any blood vessels, the cells are dependent on cytokines for the communication among themselves in the extra-cellular matrix and also for communication with the outside world. Various cytokines have been found to be able to penetrate the extra-cellular matrix and inhibit, respectively stimulate the proteoglycan synthesis. Also, the degradation of proteoglycans can be stimulated, respectively inhibited by several cytokines. In addition, some cytokines have been found which regulate the effects of the other cytokines. With respect to proteoglycan metabolism a complex cytokine network is emerging.Furthermore it is becoming increasingly clear that proteoglycans are connected to the cytokine network by their own bioactive functions. First, they possibly possess cytokine activities themselves. Second, they can function as receptors, protectors, inactivators and storage ligands for cytokines. So the proteoglycans are clearly involved in the feedback signalling from the extra-cellular matrix to the cells that are synthesizing this extra-cellular matrix. Together with agonistic or antagonistic cytokines they are involved in the regulation of proteoglycan turnover during balanced or unbalanced metabolism in normal, respectively pathological situations.     

A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9 weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

Aggrecan, aging and assembly in articular cartilage

The primary function of articular cartilage to act as a self-renewing, low frictional material that can distribute load efficiently at joints is critically dependent upon the composition and organisation of the extracellular matrix. Aggrecan is a major component of the extracellular matrix, forming high molecular weight aggregates necessary for the hydration of cartilage and to meet its weight-bearing mechanical demands. Aggregate assembly is a highly ordered process requiring the formation of a ternary complex between aggrecan, link protein and hyaluronan. There is extensive age-associated heterogeneity in the structure and molecular stoichiometry of these components in adult human articular cartilage, resulting in diverse populations of complexes with a range of stabilities that have implications for cartilage mechanobiology and integrity. Recent findings have demonstrated that aggrecan can form ligands with other matrix proteins. These findings provide new insights into mechanisms for aggregate assembly and functional protein networks in different cartilage compartments with maturation and aging.     

Molecular basis of osteoarthritis: biomechanical aspects

The unique biomechanical properties of healthy cartilage ensure that articular cartilage is able to transmit force between the joints while maintaining almost friction-free limb movement. In osteoarthritis, the biomechanical properties are compromised, but we still do not understood whether this precedes the onset of the disease or is a result of it. This review focuses on the physical changes to cartilage with age, disease, and mechanical loading, with specific reference to the increased collagen cross-linking that occurs with age (nonenzymatic glycation), and the response of chondrocytes to physiological and pathological loads. In addition, the biomechanical properties and matrix biosynthesis of cartilage from various joint surfaces of the knee and ankle are compared to elucidate reasons why the ankle is less affected by progressive osteoarthritis than the knee.     

Thrombospondins: from structure to therapeutics

Cartilage oligomeric matrix protein, also known as thrombospondin-5 (TSP-5), is an extracellular matrix protein found primarily in cartilage and musculoskeletal tissues. TSP-5 is of interest because mutations in the gene cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Both PSACH and EDM1 have a characteristic chondrocyte phenotype distinguished by giant rough endoplasmic reticulum (rER) cisternae containing TSP-5 and other extracellular matrix proteins such as type IX collagen and matrilin-3. The accumulation of proteinaceous material in the rER compromises cellular function and leads to premature chondrocyte death. Both in vitro and in vivo models have been generated with varying degrees of success to study the cellular mechanisms of the disease process. Here we review and discuss in vitro and in vivo PSACH and MED model systems and describe two transgenic mouse lines expressing human mutant TSP-5 protein. These model systems have revealed several important features of the PSACH cellular pathology: unfolded protein response activation, upregulation of apoptosis and inappropriate assembly of matrix network in the rER. Some of these models are valuable reagents that may be of use in testing therapeutic interventions. (Part of a Multiauthor Review).     

On the existence of cytokines in invertebrates

Based on the assumption that invertebrates, like vertebrates, possess factors regulating responses to infection or wounding, studies dealing with the evolution of immunity have focussed on the isolation and characterisation of putative cytokine-related molecules from invertebrates. Until recently, most of our knowledge of cytokine- and cytokine receptor-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. As such, a phylogenetic relationship between invertebrate cytokine-like molecules and vertebrate counterparts could not be convincingly demonstrated. Recent genomic sequence analyses of interleukin-1-receptor-related molecules, that is Toll-like receptors, and members of the transforming growth factor-β superfamily suggest that the innate immune system of invertebrates and vertebrates evolved independently. In addition, data from protochordates and annelids suggest that invertebrate cytokine-like molecules and vertebrate factors do not have the same evolutionary origin. We propose instead that the convergence of function of invertebrate cytokine-like molecules and vertebrate counterparts involved in innate immune defences may be based on similar lectin-like activities. Received 27 November 2000; received after revision 11 December 2000; accepted 13 December 2000     

Les tests microbiologiques pour la détermination des vitamines

Summary The growth factors for microorganisms are nothing else than the vitamines known in animal physiology. Some microorganisms, like animals, have lost the possibility of synthetising these factors, which must be added to the culture medium. The action of the vitamine is quantitative and specific. These properties enable the auxo-heterotrophic microorganism, having lost the possibility of synthesis for a determined vitamine, to be used for a quantitative determination of this factor. The Phycomyces test for thiamine (vitamine B₁) and its different practical applications are discussed.     

The role of growth hormone and insulin-like growth factors in the immune system

Growth hormone (GH) and insulin-like growth factor I (IGF-I) can modulate the development and function of the immune system. In this chapter, we present data on the expression of receptors for GH and IGFs and the in vitro and in vivo effects of these proteins. We show that expression of GH and IGFs in the immune system opens up the possibility that these proteins are not only involved in endocrine control of the immune system but can also play a role as local growth and differentiation factors (cytokines). Endocrine control of GH could be direct or mediated via endocrine or autocrine/paracrine IGF-I. In addition, GH can act as an autocrine or paracrine factor itself. Furthermore, IGF-I in the immune system has been shown to be regulated by cytokines, such as interleukin-1 and interferon-γ, alluding to a cytokine-like function of IGF-I. In addition to data on the function of GH and IGF-I in the immune system, we present new findings which imply a possible function of IGF-II and IGF-binding proteins.     

Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development

Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudopepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S₄ through S₃′ subsites. Hydrophobic and aromatic amino acids are preferred at the P₁ and P₁′ positions. Interactions at additional subsites are important in some cases, for example with cleavage of κ-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones. Received 25 May 2001; received after revision 27 August 2001; accepted 30 August 2001     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.