The microRNA-455 Null Mouse Has Memory Deficit and Increased Anxiety, Targeting Key Genes Involved in Alzheimer's Disease.

The complete molecular mechanisms underlying the pathophysiology of Alzheimer's disease (AD) remain to be elucidated. Recently, microRNA-455-3p has been identified as a circulating biomarker of early AD, with increased expression in post-mortem brain tissue of AD patients. MicroRNA-455-3p also directly targets and down-regulates APP, with the overexpression of miR-455-3p suppressing its toxic effects. Here, we show that miR-455-3p expression decreases with age in the brains of wild-type mice. We generated a miR-455 null mouse utilising CRISPR-Cas9 to explore its function further. Loss of miR-455 resulted in increased weight gain, potentially indicative of metabolic disturbances. Furthermore, performance on the novel object recognition task diminished significantly in miR-455 null mice (p = 0.004), indicating deficits in recognition memory. A slight increase in anxiety was also captured on the open field test. BACE1 and TAU were identified as new direct targets for miR-455-3p, with overexpression of miR-455-3p leading to a reduction in the expression of APP, BACE1 and TAU in neuroblastoma cells. In the hippocampus of miR-455 null mice at 14 months of age, the levels of protein for APP, BACE1 and TAU were all increased. Such findings reinforce the involvement of miR-455 in AD progression and demonstrate its action on cognitive performance.

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.

The role of microRNA-3085 in chondrocyte function.

MicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFß1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFß1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

microRNA-seq of cartilage reveals an overabundance of miR-140-3p which contains functional isomiRs.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

The function of microRNAs in cartilage and osteoarthritis.

MicroRNAs are small double-stranded RNAs, which negatively regulate gene expression and have been shown to have key roles in both chondrocyte development and cartilage homeostasis with age. Deletion of all microRNAs in chondrocytes leads to skeletal growth defects in mice, whilst deletion of specific microRNAs, e.g. miR-140, leads to premature articular cartilage degradation and increased susceptibility to posttraumatic osteoarthritis. Studies comparing microRNA expression in normal human articular cartilage compared to osteoarthritic cartilage show differential expression, but varying sample groups make interpretation difficult. MicroRNAs have been proposed as circulating biomarkers of osteoarthritis, but again, this differs amongst patient cohorts. Many micro-RNAs have been shown to have roles in chondrocyte phenotype via signalling pathways, apoptosis, autophagy and senescence. Modulating microRNAs in the joint has been shown to reduce osteoarthritis in animal models and translating this to man as a novel therapeutic strategy will be key.

The microRNA-29 family in cartilage homeostasis and osteoarthritis.

UNLABELLED: MicroRNAs have been shown to function in cartilage development and homeostasis, as well as in progression of osteoarthritis. The objective of the current study was to identify microRNAs involved in the onset or early progression of osteoarthritis and characterise their function in chondrocytes. MicroRNA expression in mouse knee joints post-DMM surgery was measured over 7 days. Expression of miR-29b-3p was increased at day 1 and regulated in the opposite direction to its potential targets. In a mouse model of cartilage injury and in end-stage human OA cartilage, the miR-29 family was also regulated. SOX9 repressed expression of miR-29a-3p and miR-29b-3p via the 29a/b1 promoter. TGFß1 decreased expression of miR-29a, b, and c (3p) in primary chondrocytes, whilst IL-1ß increased (but LPS decreased) their expression. The miR-29 family negatively regulated Smad, NFκB, and canonical WNT signalling pathways. Expression profiles revealed regulation of new WNT-related genes. Amongst these, FZD3, FZD5, DVL3, FRAT2, and CK2A2 were validated as direct targets of the miR-29 family. These data identify the miR-29 family as microRNAs acting across development and progression of OA. They are regulated by factors which are important in OA and impact on relevant signalling pathways. KEY MESSAGES: Expression of the miR-29 family is regulated in cartilage during osteoarthritis. SOX9 represses expression of the miR-29 family in chondrocytes. The miR-29 family is regulated by TGF-ß1 and IL-1 in chondrocytes. The miR-29 family negatively regulates Smad, NFκB, and canonical Wnt signalling. Several Wnt-related genes are direct targets of the miR-29 family.

Novel WWP2 ubiquitin ligase isoforms as potential prognostic markers and molecular targets in cancer.

The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFß/Smad signalling activity linked to epithelial-mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFß, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFß-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFß/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFß-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets.

Class I histone deacetylase inhibition modulates metalloproteinase expression and blocks cytokine-induced cartilage degradation.

OBJECTIVE: To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro. METHODS: A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro. RESULTS: Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression. CONCLUSION: Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

MMP-14 and MMP-2 are key metalloproteases in Dupuytren's disease fibroblast-mediated contraction.

Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.

The expression and function of microRNAs in chondrogenesis and osteoarthritis.

OBJECTIVE: To use an in vitro model of chondrogenesis to identify microRNAs (miRNAs) with a functional role in cartilage homeostasis. METHODS: The expression of miRNAs was measured in the ATDC5 cell model of chondrogenesis using microarray and was verified using quantitative reverse transcription-polymerase chain reaction. MicroRNA expression was localized by in situ hybridization. Predicted miRNA target genes were validated using 3'-untranslated region-Luc reporter plasmids containing either wild-type sequences or mutants of the miRNA target sequence. Signaling through the Smad pathway was measured using a (CAGA)(12) -Luc reporter. RESULTS: The expression of several miRNAs was regulated during chondrogenesis. These included 39 miRNAs that are coexpressed with miRNA-140 (miR-140), which is known to be involved in cartilage homeostasis and osteoarthritis (OA). Of these miRNAs, miR-455 resides within an intron of COL27A1 that encodes a cartilage collagen. When human OA cartilage was compared with cartilage obtained from patients with femoral neck fractures, the expression of both miR-140-5p and miR-455-3p was increased in OA cartilage. In situ hybridization showed miR-455-3p expression in the developing limbs of chicks and mice and in human OA cartilage. The expression of miR-455-3p was regulated by transforming growth factor ß (TGFß) ligands, and miRNA regulated TGFß signaling. ACVR2B, SMAD2, and CHRDL1 were direct targets of miR-455-3p and may mediate its functional impact on TGFß signaling. CONCLUSION: MicroRNA-455 is expressed during chondrogenesis and in adult articular cartilage, where it can regulate TGFß signaling, suppressing the Smad2/3 pathway. Diminished signaling through this pathway during the aging process and in OA chondrocytes is known to contribute to cartilage destruction. We propose that the increased expression of miR-455 in OA exacerbates this process and contributes to disease pathology.

Superoxide dismutase downregulation in osteoarthritis progression and end-stage disease.

BACKGROUND: Oxidative stress is proposed as an important factor in osteoarthritis (OA). OBJECTIVE: To investigate the expression of the three superoxide dismutase (SOD) antioxidant enzymes in OA. METHODS: SOD expression was determined by real-time PCR and immunohistochemistry using human femoral head cartilage. SOD2 expression in Dunkin-Hartley guinea pig knee articular cartilage was determined by immunohistochemistry. The DNA methylation status of the SOD2 promoter was determined using bisulphite sequencing. RNA interference was used to determine the consequence of SOD2 depletion on the levels of reactive oxygen species (ROS) using MitoSOX and collagenases, matrix metalloproteinase 1 (MMP-1) and MMP-13, gene expression. RESULTS: All three SOD were abundantly expressed in human cartilage but were markedly downregulated in end-stage OA cartilage, especially SOD2. In the Dunkin-Hartley guinea pig spontaneous OA model, SOD2 expression was decreased in the medial tibial condyle cartilage before, and after, the development of OA-like lesions. The SOD2 promoter had significant DNA methylation alterations in OA cartilage. Depletion of SOD2 in chondrocytes increased ROS but decreased collagenase expression. CONCLUSION: This is the first comprehensive expression profile of all SOD genes in cartilage and, importantly, using an animal model, it has been shown that a reduction in SOD2 is associated with the earliest stages of OA. A decrease in SOD2 was found to be associated with an increase in ROS but a reduction of collagenase gene expression, demonstrating the complexities of ROS function.

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis.

OBJECTIVE: Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). METHODS: Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. RESULTS: Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. CONCLUSION: Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

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.

Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level.

mRNA profiling is routinely used to identify microRNA targets, however, this high-throughput technology is not suitable for identifying targets regulated only at protein level. Here, we have developed and validated a novel methodology based on computational analysis of promoter sequences combined with mRNA microarray experiments to reveal transcription factors that are direct microRNA targets at the protein level. Using this approach we identified Smad3, a key transcription factor in the TGFbeta signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFbeta pathway through repression of Smad3 and that TGFbeta suppressed the accumulation of miR-140 forming a double negative feedback loop. Our findings establish a valid strategy for the discovery of microRNA targets regulated only at protein level, and we propose that additional targets could be identified by re-analysis of existing microarray datasets.

Characterization and regulation of ADAMTS-16.

The ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family includes 19 secreted proteinases in man. ADAMTS16 is a recently cloned gene expressed at high levels in fetal lung and kidney and adult brain and ovary. The ADAMTS-16 protein currently has no known function. ADAMTS16 is also expressed in human cartilage and synovium where its expression is increased in tissues from osteoarthritis patients compared to normal tissues. In this study, we ascertained that the full length ADAMTS16 mRNA was expressed in chondrocytes and cloned the appropriate cDNA. Stable over-expression of ADAMTS16 in chondrosarcoma cells led to a decrease in cell proliferation and migration, though not adhesion, as well as a decrease in the expression of matrix metalloproteinase-13 (MMP13). The transcription start point of the human ADAMTS16 gene was experimentally identified as 138 bp upstream of the translation start ATG and the basal promoter was mapped out to -1802 bp. Overexpression of Egr1 induced ADAMTS16 promoter constructs of -157/+138 or longer whilst Sp1 induced all ADAMTS16 promoter constructs. Transforming growth factor beta (TGFbeta) stimulated expression of endogenous ADAMTS16 gene expression in chondrocyte cell lines.

Degradome expression profiling in human articular cartilage.

INTRODUCTION: The molecular mechanisms underlying cartilage destruction in osteoarthritis are poorly understood. Proteolysis is a key feature in the turnover and degradation of cartilage extracellular matrix where the focus of research has been on the metzincin family of metalloproteinases. However, there is strong evidence to indicate important roles for other catalytic classes of proteases, with both extracellular and intracellular activities. The aim of this study was to profile the expression of the majority of protease genes in all catalytic classes in normal human cartilage and that from patients with osteoarthritis (OA) using a quantitative method. METHODS: Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur (NOF). Total RNA was purified, and expression of genes assayed using Taqman low-density array quantitative RT-PCR. RESULTS: A total of 538 protease genes were profiled, of which 431 were expressed in cartilage. A total of 179 genes were differentially expressed in OA versus NOF cartilage: eight aspartic proteases, 44 cysteine proteases, 76 metalloproteases, 46 serine proteases and five threonine proteases. Wilcoxon ranking as well as the LogitBoost-NR machine learning approach were used to assign significance to each gene, with the most highly ranked genes broadly similar using each method. CONCLUSIONS: This study is the most complete quantitative analysis of protease gene expression in cartilage to date. The data help give direction to future research on the specific function(s) of individual proteases or protease families in cartilage and may help to refine anti-proteolytic strategies in OA.

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.

The PRH/Hex repressor protein causes nuclear retention of Groucho/TLE co-repressors.

The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a transcription factor that functions as an important regulator of vertebrate development and many other processes in the adult including haematopoiesis. The Groucho/TLE (transducin-like enhancer) family of co-repressor proteins also regulate development and modulate the activity of many DNA-binding transcription factors during a range of diverse cellular processes including haematopoiesis. We have shown previously that PRH is a repressor of transcription in haematopoietic cells and that an Eh-1 (Engrailed homology) motif present within the N-terminal transcription repression domain of PRH mediates binding to Groucho/TLE proteins and enables co-repression. In the present study we demonstrate that PRH regulates the nuclear retention of TLE proteins during cellular fractionation. We show that transcriptional repression and the nuclear retention of TLE proteins requires PRH to bind to both TLE and DNA. In addition, we characterize a trans-dominant-negative PRH protein that inhibits wild-type PRH activity by sequestering TLE proteins to specific subnuclear domains. These results demonstrate that transcriptional repression by PRH is dependent on TLE availability and suggest that subnuclear localization of TLE plays an important role in transcriptional repression by PRH.

The regulation of matrix metalloproteinases and their inhibitors.

The matrix metalloproteinases (MMP) are a family of 23 enzymes in man. These enzymes were originally described as cleaving extracellular matrix (ECM) substrates with a predominant role in ECM homeostasis, but it is now clear that they have much wider functionality. Control over MMP and/or tissue inhibitor of metalloproteinases (TIMP) activity in vivo occurs at different levels and involves factors such as regulation of gene expression, activation of zymogens and inhibition of active enzymes by specific inhibitors. Whilst these enzymes and inhibitors have clear roles in physiological tissue turnover and homeostasis, if control of their expression or activity is lost, they contribute to a number of pathologies including e.g. cancer, arthritis and cardiovascular disease. The expression of many MMPs and TIMPs is regulated at the level of transcription by a variety of growth factors, cytokines and chemokines, though post-transcriptional pathways may contribute to this regulation in specific cases. The contribution of epigenetic modifications has also been uncovered in recent years. The promoter regions of many of these genes have been, at least partly, characterised including the role of identified single nucleotide polymorphisms. This article aims to review current knowledge across these gene families and use a bioinformatic approach to fill the gaps where no functional data are available.