35 research outputs found
Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1–mediated endocytosis
No full textMatrix protease activity is fundamental to developmental tissue patterning and remains influential in adult homeostasis. In cartilage, the principal matrix proteoglycan is aggrecan, the protease-mediated catabolism of which defines arthritis; however, the pathophysiologic mechanisms that drive aberrant aggrecanolytic activity remain unclear. Human ciliopathies exhibit altered matrix, which has been proposed to be the result of dysregulated hedgehog signaling that is tuned within the primary cilium. Here, we report that disruption of intraflagellar transport protein 88 (IFT88), a core ciliary trafficking protein, increases chondrocyte aggrecanase activity in vitro. We find that the receptor for protease endocytosis in chondrocytes, LDL receptor–related protein 1 (LRP-1), is unevenly distributed over the cell membrane, often concentrated at the site of cilia assembly. Hypomorphic mutation of IFT88 disturbs this apparent hot spot for protease uptake, increases receptor shedding, and results in a reduced rate of protease clearance from the extracellular space. We propose that IFT88 and/or the cilium regulates the extracellular remodeling of matrix—independently of Hedgehog regulation—by enabling rapid LRP-1–mediated endocytosis of proteases, potentially by supporting the creation of a ciliary pocket. This result highlights new roles for the cilium’s machinery in matrix turnover and LRP-1 function, with potential relevance in a range of diseases.—Coveney, C. R., Collins, I., Mc Fie, M., Chanalaris, A., Yamamoto, K., Wann, A. K. T. Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1–mediated endocytosis
Array CGH analysis at 60kb resolution of CML samples at advanced stage of disease
No full textIn spite of the universal presence of the BCR/ABL1 fusion gene, chronic myelogenous leukemia (CML) shows remarkable clinical and genetic diversity. The consequences of der(9)t(9;22) chromosome deletions, associated with poor survival, as well as the mechanism behind their formation remain unclear, as does our understanding of the molecular events driving the disease evolution. The presence of these deletions fuelled the expectations that cryptic genome-wide aberrations may be accountable for the disease progression. Following a comprehensive BAC aCGH analysis of 48 CML samples (Brazma et al., Genes, Chromosomes & Cancer, 2007 in press) we report high-resolution oligo-nucleotide array study of a further 30 CML accelerated/ blast phase samples. We were unable to confirm the high frequency of particular single BAC imbalances (CNVs), reported both by ourselves and others, possibly due to the manufacturer’s array selection strategy. Never-the-less some of the CNVs and a wealth of new imbalances were obtained at 60kb resolution. It was possible to build a precise map of the amplicon affecting the sequences flanking the 3' ABL1 breakpoint site, which include the LAMC3 and NUP214 genes. The presence of this amplicon was associated with therapy resistance. When assessed, at a resolution of 60 kb, the deletions of the regions flanking the ABL1/BCR breakpoint showed novel features:
1. the genome loss affects preferentially both flanking sites as seen in 5 of the 6 ‘deleted’ samples and 2. the 120kb deletion identified is the smallest recorded so far. Most of the major cytogenetic features of the samples were confirmed and a number of cryptic genome imbalances were detected, from 120kb to 10Mb in size, involving regions rich in genes, some already implicated in the pathogenesis of CML. Finally, recurrent micro aberrations of several adjacent oligo-nucleotides affecting non-coding sequences were detected in as many as 2/3 of the samples
Hypertrophic effects of urocortin homologous peptides are mediated via activation of the Akt pathway
No full textThe UCN homologues SCP and SRP bind specifically to the CRFR2 receptor, whereas UCN binds to both CRFR1 and CRFR2. We have previously demonstrated that all three peptides are cardioprotective, and both the Akt and MAPK p42/44 pathways are essential for this effect. Here we tested the hypertrophic effects of these peptides. We examined the effects of the peptides on cell area, protein synthesis, and induction of the natriuretic peptides ANP and BNP. All three peptides were able to increase all the markers of hypertrophy examined, with SCP being the most potent of the three, followed by UCN and SRP last. In addition, we provide a mechanism of action for the three peptides and show that Akt phosphorylation is important for their hypertrophic action, whereas MAPK p42/44 is not involved in this effect
Ciliary proteins specify the cell inflammatory response by tuning NFκB signaling, independently of primary cilia
No full textComplex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting these pathways as therapeutic targets. Primary cilia are sub-cellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviors, changing cytosolic NFκB translocation dynamics, but leaving MAPK unaffected. RNAseq analysis indicates IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. By microscopy, we find altered NFκB dynamics are independent to assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits the inflammatory responses in the non-ciliated macrophage. We propose ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme, to tune cytoplasmic NFκB signalling, and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation
2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.
No full textWe studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation
Cardioprotection mediated by urocortin is dependent on PKCepsilon activation
No full textUrocortin (Ucn) is an endogenous cardioprotective agent that protects against the damaging effects of ischemia and reperfusion injury in vitro and in vivo. We have found that the mechanism of action of Ucn involves both acute activation of specific target molecules, and using Affymetrix (Santa Clara, CA) gene chip technology, altered gene expression of different end effector molecules. Here, from our gene chip data, we show that after a 24 h exposure to Ucn, there was a specific increase in mRNA and protein levels of the protein kinase C epsilon (PKC?) isozyme in primary rat cardiomyocytes compared with untreated cells and in the Langendorff perfused ex vivo heart. Furthermore, a short 10 min exposure of these cells to Ucn caused a specific translocation/activation of PKC? in vitro and in the Langendorff perfused ex vivo heart. The importance of the PKC? isozyme in cardioprotection and its relationship to cardioprotection produced by Ucn was assessed using PKC?-specific inhibitor peptides. The inhibitor peptide, when introduced into cardiomyocytes, caused an increase in apoptotic cell death compared with control peptide after ischemia and reperfusion. When the inhibitor peptide was present with Ucn, the cardioprotective effect of Ucn was lost. This loss of cardioprotection by Ucn was also seen in whole hearts from PKC? knockout mice. These findings indicate that the cardioprotective effect of Ucn is dependent upon PKC?
Differential regulation of FGFR3 controls FGFR1:FGFR3 balance in chondrocytes after cartilage injury
No full textRelease of FGF-2 from the pericelluar matrix of cartilage upon injury is an important early response of the tissue to injury. Even though FGF-2 knockout mice have substantially increased cartilage degradation in spontaneous and surgically induced models of osteoarthritis (OA), the in vitro literature is divided about the role of FGF-2 in articular cartilage. It is currently unclear whether FGF-2 is chondroprotective by promoting anabolic effects; or destructive through promotion of catabolic responses. One theory is that FGF-2’s opposing effects depend on which receptor it acts through. Knockout studies of FGFRs in mice have shed some light on this theory. FGFR1 conditional knockout mice are protected from developing disease while FGFR3 knockout mice have worse disease, suggesting FGFR1 mediates pro-catabolic effects and FGFR3 mediates chondroprotective effects of FGF-2. As FGFR ratios have been shown to be dysregulated in OA, we studied how receptor ratios were affected by cartilage injury, tissue culture and hypoxia
ADAMTS and ADAM metalloproteinases in osteoarthritis – looking beyond the ‘usual suspects’
No full textINTRODUCTION: Matrix metalloproteinases (MMPs) and 'aggrecanase' a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are well established to play key roles in osteoarthritis (OA) through degradation of extracellular matrix (ECM) type II collagen and aggrecan, and are thus potential targets for development of OA therapies. OBJECTIVE: This paper aims to provide a comprehensive review of the expression and potential roles of other, lesser-known ADAMTSs and related adamalysins (or a disintegrin and metalloproteinases (ADAMs)) in cartilage, with a view to identifying potentially protective or homeostatic metalloproteinases in the joint and informing consequent selective inhibitor design. DESIGN: A comprehensive literature search was performed using PubMed terms 'osteoarthritis' and 'ADAMTS' or 'ADAM'. RESULTS: Several ADAMTSs and ADAMs were identified as having reportedly increased expression in OA. These include enzymes likely to play roles in cartilage matrix anabolism (e.g., the procollagen N-proteinases ADAMTS-2, ADAMTS-3 and ADAMTS-14), chondrocyte differentiation and proliferation (e.g., ADAM9, ADAM10, ADAM12), as well as enzymes contributing to cartilage catabolism (e.g., Cartilage oligomeric protein (COMP)-degrading ADAMTS-7 and ADAMTS-12). CONCLUSIONS: In addition to the well-characterised MMPs, ADAMTS-4 and ADAMTS-5, many other ADAMTSs and ADAMs are expressed in cartilage and several show significantly altered expression in OA. Studies aimed at elucidating the pathophysiological roles of these enzymes in cartilage will contribute to our understanding of OA pathogenesis and enable design of targeted inhibitors that effectively target metalloproteinase-mediated cartilage degradation while sparing cartilage repair pathways
TNFAIP6 and SAA1 mRNA levels are elevated in peripheral blood cells following destabilising knee injuries
No full textPurpose: Acute destabilising injury to the joint is a considerable risk for the development of osteoarthritis (OA); about 50% of individuals with such injuries will develop OA within 5 to 10 years. It is currently impossible to predict whether an individual with an acute destabilising joint injury will develop OA or not. Our previous work in mice has revealed a number of highly mechanosensitive genes/pathways that are regulated early upon surgical destabilization of the medial meniscus (DMM). As many of the genes identified were ubiquitously expressed, inflammatory response genes, we asked whether they were also regulated in the cellular compartment of the peripheral blood. Finally, we examined whether genes that were regulated in the mouse blood upon DMM were also regulated in the peripheral blood of patients who had recently sustained a destabilising knee injury
