Andrew E. Parker

Metalloproteinases: their role in arthritis and potential as therapeutic targets

Irreversible degradation of articular cartilage is a major feature of the arthritides, and its prevention is a therapeutic goal which has been difficult to achieve. Enzymes from the matrix metalloproteinase and ADAMTS (a disintegrin, a metalloproteinase, and thrombospondin motif) families are key mediators of cartilage extracellular matrix destruction. Inhibition of metalloproteinase activity is therefore a conceptually attractive therapeutic strategy, although clinical efficacy has not yet been demonstrated. This review outlines the biology behind metalloproteinases as drug targets in the arthritides, and poses important questions for the future design of such therapies.

HIF-VEGF Pathways Are Critical for Chronic Otitis Media in Junbo and Jeff Mouse Mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF–mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF–mediated pathways, and we conclude that targeting molecules in HIF–VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice.

The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.

Nurr1 dependent regulation of pro-inflammatory mediators in immortalised synovial fibroblasts

BackgroundNurr1 is an orphan member of the nuclear receptor superfamily; these orphan receptors are a group for which a ligand has yet to be identified. Nurr1 has been shown to regulate the expression of a small number of genes as a monomeric, constitutively active receptor. These Nurr1 regulated genes are primarily associated with dopamine cell maturation and survival. However, previous reports have shown an increased expression of Nurr1 in the synovium of patients with rheumatoid arthritis (RA) suggesting a pro-inflammatory role for Nurr1 in RA. In this study we investigate the potential pro-inflammatory role of Nurr1 by monitoring Nurr1 dependent gene expression in an immortalised synoviocyte cell line, K4IM.MethodsWe overexpressed the wild type and a dominant negative form of the orphan nuclear receptor Nurr1, in a model synoviocyte cell line. Using the Affymetrix HG-U133 Genechips we demonstrate the effects on the transcriptome by the receptor. Further evidence of gene expression change was demonstrated using quantitative RT-PCR and ELISA analysis.ResultsWe show that Nurr1 regulates transcription of a small number of genes for pro-inflammatory modulators of which the most significant is interleukin-8 (IL-8). We also demonstrate increased synthesis and secretion of IL-8 further supporting a role for Nurr1 in inflammatory signalling pathways.ConclusionUsing microarray analysis we show that elevated levels of Nurr1 leads to increased gene expression of pro-inflammatory genes: IL-8, Amphiregulin and Kit ligand in a model cell line. This data provides further evidence for an additional role for Nurr1 in inflammation and may play a role in the pathogenesis of rheumatoid arthritis.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1)-mediated Endocytic Clearance of a Disintegrin and Metalloproteinase with Thrombospondin Motifs-4 (ADAMTS-4) FUNCTIONAL DIFFERENCES OF NON-CATALYTIC DOMAINS OF ADAMTS-4 AND ADAMTS-5 IN LRP1 BINDING

Background: LRP1 is an endocytic receptor of ADAMTS-5 (aggrecanase 2) in cartilage. Results: ADAMTS-4 (aggrecanase 1) is also internalized via LRP1 but at a slower rate than ADAMTS-5. Conclusion: LRP1 differently regulates the extracellular activities of the two key aggrecanases in cartilage. Significance: LRP1 is a major traffic controller of the two aggrecanases. Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

Expression and function of matrix metalloproteinase (MMP)-28.

Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis. In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA. These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.

MMP28 gene expression is regulated by Sp1 transcription factor acetylation

MMP-28 (epilysin) is a recently cloned member of the MMP (matrix metalloproteinase) family. It is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues, as well as a number of carcinomas. The MMP28 promoter has previously been cloned and characterized identifying a conserved GT-box that binds Sp1/Sp3 (specificity proteins 1 and 3) proteins and is essential for the basal expression of the gene. The present study demonstrates that MMP28 expression is induced by HDAC (histone deacetylase) inhibitors and that this effect is mediated through the GT-box. Transient transfection assays have shown that the induction of MMP28 expression by the HDAC inhibitior TSA (trichostatin A) is mediated via Sp1 at the GT-box. Immunoprecipitation experiments have shown that the acetylation of Sp1 and Sp3 is increased by TSA treatment; however, no effect on DNA binding was observed. Histone acetyltransferases such as p300 and P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] increased induction of the MMP28 promoter by Sp1. Knockdown of HDAC1 using siRNA (small interfering RNA) also induces the MMP28 promoter. Oligonucleotide pulldown identified STRAP (serine/threonine kinase receptor-associated protein) as a further protein recruited to the MMP28 promoter and acting functionally with Sp1.

Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment

Objective Aggrecan is a critical component of cartilage extracellular matrix. Several members of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1β (IL-1β) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated. Methods MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1β in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1β or meniscotibial ligament transaction was carried out in MMP17 null mice. Results The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1β mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1β into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17. Conclusion These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.

Localization of the cis-enhancer element for mouse type X collagen expression in hypertrophic chondrocytes in vivo.

The type X collagen gene (Col10a1) is a specific molecular marker of hypertrophic chondrocytes during endochondral bone formation. Mutations in human COL10A1 and altered chondrocyte hypertrophy have been associated with multiple skeletal disorders. However, until recently, the cis‐enhancer element that specifies Col10a1 expression in hypertrophic chondrocytes in vivo has remained unidentified. Previously, we and others have shown that the Col10a1 distal promoter (−4.4 to −3.8 kb) may harbor a critical enhancer that mediates its tissue specificity in transgenic mice studies. Here, we report further localization of the cis‐enhancer element within this Col10a1 distal promoter by using a similar transgenic mouse approach. We identify a 150‐bp Col10a1 promoter element (−4296 to −4147 bp) that is sufficient to direct its tissue‐specific expression in vivo. In silico analysis identified several putative transcription factor binding sites including two potential activator protein‐1 (AP‐1) sites within its 5′‐ and 3′‐ends (−4276 to −4243 and −4166 to −4152 bp), respectively. Interestingly, transgenic mice using a reporter construct deleted for these two AP‐1 elements still showed tissue‐specific reporter activity. EMSAs using oligonucleotide probes derived from this region and MCT cell nuclear extracts identified DNA/protein complexes that were enriched from cells stimulated to hypertrophy. Moreover, these elements mediated increased reporter activity on transfection into MCT cells. These data define a 90‐bp cis‐enhancer required for tissue‐specific Col10a1 expression in vivo and putative DNA/protein complexes that contribute to the regulation of chondrocyte hypertrophy. This work will enable us to identify candidate transcription factors essential both for skeletal development and for the pathogenesis of skeletal disorders.

The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

ABSTRACT Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. Summary: The ENU-derived mouse mutant goya, reveals, for the first time, multiple roles of MAP3K1 in cochlear development and correct auditory function.