The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE: To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS: Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS: We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) modulates key biological pathways associated with OA disease pathology.

OBJECTIVE: To examine the role of mitogen-activated protein kinase-activated protein kinase 2 (MK2) in mediating the cellular response to pro-inflammatory cytokines in human primary osteoarthritis (OA) chondrocytes. METHODS: Delivery of a dominant negative MK2 was achieved in HeLa cells by adenoviral infection. Cellular heat shock protein (HSP27) activity was determined using a Bioplex assay. Primary OA chondrocytes were isolated by collagenase digestion of human articular cartilage. Phosphorylated MK2 was detected by immunoblotting and immunohistology. Transfection of primary chondrocytes with siRNA was achieved using cationic lipid and gene expression determined by real-time polymerase chain reaction. Production of prostaglandin E2 (PGE2) and matrixmetalloproteases (MMPs) was measured by enzyme-linked immunosorbent assay. RESULTS: Over-expression of a dominant negative MK2 inhibited HSP27 phosphorylation and significantly reduced both interleukin 1 (IL-1)beta and tumour necrosis factor (TNF)-alpha mediated release of PGE2 in HeLa cells over a 24h period. Phosphorylated MK2 was detected in OA articular cartilage and in isolated primary OA chondrocytes, where it was induced by IL-1beta. Transfection of OA chondrocytes with MK2 siRNA antisense significantly reduced both basal and IL-1beta induced PGE2 release. siRNA mediated MK2 knockdown also significantly reduced both basal and IL-1beta induced MMP13 expression and MMP13 and MMP3 protein release but had no effect on MMP1. CONCLUSIONS: Our data reveal that MK2 is active in OA human articular cartilage and in isolated primary human chondrocytes and that MK2 mediates the release of PGE2, MMP3 and MMP13. These findings suggest a role for MK2 in contributing to OA algesia and OA joint structural deterioration by mediating the downstream effects of p38 activation on PGE2 release and the expression and release of catabolic proteases.

The structure of cell-adhesion molecules.

In recent years, following the identification and molecular cloning of many key adhesion molecules, the three-dimensional structures of some of the domains that mediate adhesive interactions have been determined. This review discusses how these studies have helped explain the unique functional roles of the different families of adhesion molecules.

Production of recombinant soluble human integrin alpha4beta1.

Integrin alpha4beta1 is a major leukocyte adhesion receptor that is a key target for the development of anti-inflammatory therapeutics. With the dual long-term goals of developing a reagent for use in high-throughput inhibitor screening assays and for crystallisation trials and subsequent structure determination, we have generated a recombinant soluble alpha4beta1 receptor. Both subunits were truncated prior to the transmembrane domains by site-directed mutagenesis and expressed using baculovirus infection of insect cells. The molecular weights of the recombinant subunits were as expected for post-translationally unmodified protein. In addition, as observed for the native subunit, a proportion of the alpha4 subunit was proteolytically processed into two fragments. ELISA and solid phase ligand-binding assays were performed to investigate the folding and functionality of the soluble integrin. The data suggest that the receptor was correctly folded and that it bound recombinant ligands with similar kinetics to the native molecule.

Differential expression of e-cadherin in normal, metaplastic and dysplastic esophageal mucosa - a putative biomarker.

Barrett's mucosa is a potentially premalignant columnar lined metaplastic epithelium in the oesophagus about which little is known regarding maintenance of cell adhesion. In this regard E-Cadherin (E-cad) is a 120 kDa polypeptide present in all epithelial tissues and it is the prime mediator of cell-cell interactions. An immunohistochemical technique utilised the monoclonal antibody HECD-1 to evaluate E-cad expression in microwave treated, paraffin embedded sections from 120 patients. The phenotype of the specimens from the patients were metaplastic [41] and dysplastic mucosa [24], invasive squamous carcinoma [15] and adenocarcinoma [18] and corresponding metastatic lesions [10] as well as 12 specimens of normal oesophageal mucosae. Surface expression was high in non-metaplastic tissue and in non-dysplastic metaplasia but was reduced extensively in dysplasia. In addition in squamous carcinoma and adenocarcinoma E-cad was characteristically expressed in the cytoplasm and this was associated with poor morphological differentiation. SDS-PAGE gel protein analysis revealed the characteristic 120 kDa sized protein in normal squamous oesophageal tissue. In Barrett's metaplastic tissue and carcinoma, however, stronger bands at 108 kDa and 45 kDa were also present, suggesting either decreased glycosylation or a truncated protein in the metaplastic and neoplastic tissue, respectively. In conclusion changes in E-cad immunoreactivity and cellular localisation occur in premalignant metaplastic epithelium of the oesophagus. Further studies are in progress to evaluate whether the abnormal E-cad protein reflects both loss of function and may be used as a biomarker in pre-malignant lesions.

The orphan G-protein coupled receptor RDC1: evidence for a role in chondrocyte hypertrophy and articular cartilage matrix turnover.

OBJECTIVE: RDC1 is a class A orphan G-protein coupled receptor of unknown function. The purpose of this study was to identify compound RDC1 agonists and use these as tools to determine the effect of RDC1 activation in human chondrocytes and cartilage explant tissue. METHODS: Computational chemistry was employed to build a homology model of the RDC1 receptor. A virtual screen of in-house compounds was then performed and positive hits screened for their ability to invoke a Ca2+ response in a recombinant RDC1 HEK293 cell line, as measured by FLIPR. The effect of RDC1 activation on human chondrocytes and cartilage explant gene expression was determined by quantitative real-time polymerase chain reaction (PCR), and these effects validated as being mediated by RDC1 using siRNA antisense. RESULTS: Tissue expression profiling demonstrated that RDC1 expression was predominant in cartilage tissue. Treatment of human primary chondrocytes with RDC1 agonist induced a Ca2+ response, suggesting the receptor is active in this tissue type. Treatment for 24h with RDC1 agonist led to altered expression of a number of genes associated with chondrocyte hypertrophy and increased matrix degradation in human primary chondrocytes, and elevated total matrix metalloproteinase (MMP) activity in cartilage explant. Transfection with RDC1 siRNA caused a >90% reduction in human primary chondrocyte RDC1 expression and significantly reduced the impact of RDC1 agonist on the previously identified RDC1-regulated genes. CONCLUSIONS: RDC1 activation in human chondrocytes and cartilage explant leads to changes in gene expression and activity associated with chondrocyte hypertrophy, angiogenesis and increased matrix degradation, suggesting signalling via the RDC1 receptor may play an important role in the early development of osteoarthritis (OA).

Integrin adhesion receptors: structure, function and implications for biomedicine.

Over the past decade, multi-disciplinary approaches have led to the discovery and characterization of several classes of adhesion molecules. Under normal conditions, these molecules provide support for cells, regulate cell migration and contain information that cells use when sensing their environment. In disease, adhesive function is frequently compromised and results in tissue disorder, aberrant cell migration and dysregulation of signalling pathways. The integrins are a major family of adhesion receptors produced by most cell types and are a means by which the cell senses its immediate environment and responds to changes in extracellular matrix composition. Recent years have seen major advances in our understanding of integrin-ligand interactions, and have revealed a structurally dynamic family of receptors capable of translating information into and out of the cell.

Mechanisms of VCAM-1 and fibronectin binding to integrin alpha 4 beta 1: implications for integrin function and rational drug design.

Integrin alpha 4 beta 1 can mediate both cell-cell and cell-extracellular matrix adhesion by binding to either fibronectin or vascular cell adhesion molecule 1 (VCAM-1). Both interactions are important for extravasation of leukocytes from the blood implying that rationally designed inhibitors of alpha 4 beta 1 function may be useful for treating a various inflammatory conditions. The mechanisms of ligand binding by alpha 4 beta 1 are complicated by the fact that alternative splicing can generate different isoforms of the receptor-binding domains in both fibronectin and VCAM-1. Therefore, in addition to developing alpha 4 beta 1 antagonists, we have also been interested in identifying isoform-specific functions. Recombinant ligand variants have been tested in adhesion and direct receptor-binding assays and each molecule was found to have a different inherent affinity for alpha 4 beta 1 that endows them with different adhesive activities. This suggests that alternative splicing may regulate alpha 4 beta 1-dependent motility in vivo. The initial strategy that we have adopted to develop alpha 4 beta 1 inhibitors has been to identify key amino acid residues and peptide sequences participating in the receptor-ligand binding event and to use this information to generate synthetic mimetics. Three active sites have been identified in fibronectin by testing truncated proteins, expressing recombinant fragments and screening synthetic peptides. Two of these sites employ versions of a novel integrin-binding motif, LDVP/IDAP. A key active site in VCAM-1 has been identified by similar approaches as the related sequence IDSP. Since IDSP-like sequences are probably used by other integrin-binding immunoglobulins, derivatives of these peptides may turn out to be the forerunners of a new generation of therapeutic agents with multiple applications.

Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.

The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.

Analysis of ligand-induced and ligand-attenuated epitopes on the leukocyte integrin alpha4beta1: VCAM-1, mucosal addressin cell adhesion molecule-1, and fibronectin induce distinct conformational changes.

The leukocyte integrin alpha4beta1 is a receptor for both cell surface ligands (VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix components (fibronectin). Through regulated interactions with these molecules, alpha4beta1 mediates leukocyte migration from the vasculature at sites of inflammation. Regulation of integrin activity plays a key role in controlling leukocyte-adhesive events and appears to be partly determined by changes in integrin conformation. Several mAbs that recognize ligand-induced binding site epitopes on integrins have been characterized, and a subset of these mAbs are capable of stimulating integrin-ligand binding. Conversely, some mAbs recognize epitopes that are attenuated by ligand engagement and allosterically inhibit ligand binding. To gain insight into ligand-specific effects on integrin conformation, we have examined the ability of different ligands to modulate the binding of four distinct classes (A, B1, B2, and C) of anti-alpha4 Abs to alpha4beta1. VCAM-1 attenuated B (antifunctional) class epitopes via an allosteric mechanism and also allosterically inhibited the binding of the function-blocking anti-beta1 mAb 13. Additional alpha4beta1 ligands (fibronectin fragments, MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding; however, the epitopes of the class B anti-alpha4 mAbs were attenuated by the fibronectin fragments, but not by MAdCAM-1 or the CS1 peptide. Of the two anti-alpha4 class A mAbs examined, one recognized an epitope that was induced uniquely by VCAM-1. Taken together, these data suggest that overlapping but distinct binding mechanisms exist for different alpha4beta1 ligands and that distinct conformational changes are induced upon integrin engagement by different ligands.

A dominant negative form of IKK2 prevents suppression of apoptosis by the peroxisome proliferator nafenopin.

Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocyte cultures, PPs suppress spontaneous apoptosis and that induced by a number of pro-apoptotic stimuli such as transforming growth factor-beta(1). Tumour necrosis factor alpha (TNF-alpha) and the transcription factor NFkappaB have been implicated in the mode of action of PPs. TNF-alpha signalling to NFkappaB is thought to be responsible for many of the effects elicited by this cytokine. NFkappaB regulates gene expression in immunity, stress responses and the inhibition of apoptosis. Activation of NFkappaB requires the successive action of NFkappaB-inducing kinase and the phosphorylation of NFkappaB inhibitory proteins (IkappaB) by an IkappaB kinase (IKK) complex. The IKK2 subunit of IkappaB kinase is thought to be essential for NFkappaB activation and prevention of apoptosis. To determine whether IKK2 plays a role in the suppression of apoptosis by PPs, we expressed a dominant negative form of IKK2 (IKK2dn) in primary rat hepatocyte cultures. Infection with an adenovirus construct expressing IKK2dn caused apoptosis in control primary rat hepatocytes in the absence of exogenous TNF-alpha. Moreover, IKK2dn-induced apoptosis could not be rescued by addition of TNF-alpha or the peroxisome proliferator nafenopin. These results demonstrate a requirement for intracellular signalling pathways mediated by IKK2 in the suppression of apoptosis by the PP class of hepatocarcinogens.

Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin alpha 4 beta 1.

The integrin receptor alpha 4 beta 1 binds to two different ligands, the extracellular matrix glycoprotein fibronectin and the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1). Using probes derived from each ligand and a variety of cell adhesion and ligand-receptor binding assays, we have investigated the relationship between the mechanisms of fibronectin and VCAM-1 interaction with alpha 4 beta 1. CS1 peptide, which represents the dominant active site from the HepII/IIICS recognition domain in fibronectin, was found to inhibit VCAM-1-dependent adhesion in three different assays: MOLT-4 T lymphoblastic leukaemia cell attachment to immobilized recombinant soluble VCAM-1 (rsVCAM-1), MOLT-4 cell attachment to monolayers of VCAM-1-transfected COS-1 cells, and A375-SM melanoma cell spreading on immobilized rs VCAM-1. Half-maximal inhibition required CS1 concentrations of 1.7-3.0 mg/ml, some 3-7-fold higher than that needed to autoinhibit adhesion to CS1-IgG conjugate. Using a more sensitive solid-phase receptor-ligand binding assay, CS1 was found to be a potent inhibitor of the binding of rsVCAM-1 to alpha 4 beta 1 (half-maximal inhibition at 13 micrograms/ml). In agreement with cell-based assays, severalfold lower concentrations of CS1 were required to inhibit binding of recombinant HepII/IIICS region of fibronectin (half-maximal inhibition at 3 micrograms/ml). VCAM-1-alpha 4 beta 1 binding was blocked not only by CS1 peptide but also by the recombinant HepII/IIICS region of fibronectin. Kinetic analysis of CS1 inhibition of VCAM-1 binding revealed that it was directly competitive in nature, indicating that VCAM-1 and fibronectin recognize either identical or spatially overlapping binding sites on alpha 4 beta 1. The implications of these results for the future design of VCAM-1 antagonists are discussed.

Alpha4 integrin binding interfaces on VCAM-1 and MAdCAM-1. Integrin binding footprints identify accessory binding sites that play a role in integrin specificity.

Integrins are a family of heterodimeric adhesion receptors that mediate cellular interactions with a range of matrix components and cell surface proteins. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins (alpha4beta1 and alpha4beta7). A related CAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is recognized by alpha4beta7 but is a poor ligand for alpha4beta1. Previous studies have revealed that all alpha4 integrin-ligand interactions are dependent on a key acidic ligand motif centered on the CAM domain 1 C-D loop region. By generating VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell adhesion assays we have found that alpha4beta1 binds to the MAdCAM-1 adhesion motif when present in VCAM-1, but not when the VCAM-1 motif was present in MAdCAM-1, suggesting that this region does not contain all of the information necessary to determine integrin binding specificity. To characterize integrin-CAM specificity further we measured alpha4beta1 and alpha4beta7 binding to a comprehensive set of mutant VCAM-1 constructs containing amino acid substitutions within the predicted integrin adhesion face. These data revealed the presence of key "regulatory residues" adjacent to integrin contact sites and an important difference in the "footprint" of alpha4beta1 and alpha4beta7 that was associated with an accessory binding site located in VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and sequence and when mutated abolishes integrin binding activity.

Identification of a key integrin-binding sequence in VCAM-1 homologous to the LDV active site in fibronectin.

The integrin adhesion receptor alpha 4 beta 1 binds two ligands, the extracellular matrix glycoprotein fibronectin and the immunoglobulin superfamily member VCAM-1. Ligand-binding sites are contained with the HepII/IIICS domain of fibronectin, and within the homologous immunoglobulin domains 1 and 4 of VCAM-1. Previous studies have shown that the binding of each ligand to alpha 4 beta 1 is mutually exclusive, suggesting that they may employ similar mechanisms to bind receptor. Fibronectin contains at least three distinct peptide sequences that are active sites for alpha 4 beta 1 binding, two homologous sequences Leu-Asp-Val-Pro (LDVP) and Ile-Asp-Ala-Pro (IDAP), and a third related to Arg-Gly-Asp (RGD). Using a combination of site-directed mutagenesis and synthetic peptide approaches in conjunction with VCAM-1-dependent cell adhesion assays, we now report the identification of a key alpha 4 beta 1-binding sequence in both domains 1 and 4 of VCAM-1 as the tetrapeptide Ile-Asp-Ser-Pro (IDSP). Mutagenesis studies also suggest that an additional sequence in domain 1, KLEK, participates in receptor binding. Since IDSP is homologous to the LDVP and IDAP fibronectin peptides, this therefore provides a molecular explanation for the promiscuity of ligand binding by alpha 4 beta 1 and has implications for the design of synthetic VCAM-1 antagonists. The extrapolation of these findings to other integrin-binding immunoglobulin ligands is also discussed.