SummaryObjectivesJoint degeneration in osteoarthritis (OA) is characterised by damage and loss of articular cartilage. The pattern of loss is consistent with damage occurring only where the mechanical loading is high. We have investigated using RNA-sequencing (RNA-seq) and systems analyses the changes that occur in damaged OA cartilage by comparing it with intact cartilage from the same joint.MethodsCartilage was obtained from eight OA patients undergoing total knee replacement. RNA was extracted from cartilage on the damaged distal medial condyle (DMC) and the intact posterior lateral condyle (PLC). RNA-seq was performed to identify differentially expressed genes (DEGs) and systems analyses applied to identify dysregulated pathways.ResultsIn the damaged OA cartilage, there was decreased expression of chondrogenic genes SOX9, SOX6, COL11A2, COL9A1/2/3, ACAN and HAPLN1; increases in non-chondrogenic genes COL1A1, COMP and FN1; an altered pattern of secreted proteinase expression; but no expression of major inflammatory cytokines. Systems analyses by PhenomeExpress revealed significant sub-networks of DEGs including mitotic cell cycle, Wnt signalling, apoptosis and matrix organisation that were influenced by a core of altered transcription factors (TFs), FOSL1, AHR, E2F1 and FOXM1.ConclusionsGene expression changes in damaged cartilage suggested a signature non-chondrogenic response of altered matrix protein and secreted proteinase expression. There was evidence of a damage response in this late OA cartilage, which surprisingly showed features detected experimentally in the early response of cartilage to mechanical overload. PhenomeExpress analysis identified a hub of DEGs linked by a core of four differentially regulated TFs.
While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3-kinase C2 (PI3K-C2) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2-AKT survival pathway. ITSN associated with PI3K-C2 on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2 activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2 and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.Intersectin (ITSN) is a modular scaffold with multiple protein interaction domains that is conserved among metazoa. At the amino terminus are two Eps15 homology (EH) domains that bind NPF motifs on proteins such as epsin (36). The EH domains are followed by a coiled-coil domain that enables ITSN to homo-and heterodimerize with proteins such as Eps15 (24). The carboxy terminus consists of five Src homology 3 (SH3) domains that interact with Pro-rich motifs on a variety of proteins, several of which are involved in regulating endocytosis. Indeed, a subset of ITSNЈs SH3 domains are potent inhibitors of clathrin-coated pit formation (26). Recent studies on the Drosophila melanogaster ortholog of ITSN, Dap160, indicate that this scaffold functions as a stabilizing or recruitment factor for components of the clathrin-coated pit (14, 17). The loss of Dap160 function results in fewer coated vesicles, as well as enlarged vesicles, indicating that ITSN functions in both the formation and maturation of endocytic vesicles. Consistent with this role in Drosophila, silencing ITSN expression in mammalian cells results in defects in epidermal growth factor receptor (EGFR) internalization (18).Although ITSN is clearly linked with endocytosis, increasing evidence suggests that ITSNЈs role within the cell is not limited to this process (1,10,18,19,(27)(28)(29)32). The cloning of mammalian ITSN revealed a longer, spliced product containing a guanine nucleotide exchange factor domain specific for Cdc42 (10, 27). Through the EH domains, ITSN activates a Jun Nterminal protein kinase-dependent pathway that cooper...
Receptor tyrosine kinases (RTKs) are critical for normal cell growth, differentiation, and development, but they contribute to various pathological conditions when disrupted. Activation of RTKs stimulates a plethora of pathways, including the ubiquitylation and endocytosis of the receptor itself. Although endocytosis terminates RTK signaling, it has emerged as a requisite step in RTK activation of signaling pathways. We have discovered that the endocytic scaffolding protein intersectin (ITSN) cooperated with epidermal growth factor receptor (EGFR) in the regulation of cell growth and signaling. However, a biochemical link between ITSN and EGFR was not defined. In this study, we demonstrate that ITSN is a scaffold for the E3 ubiquitin ligase Cbl. ITSN forms a complex with Cbl in vivo mediated by the Src homology (SH) 3 domains binding to the Pro-rich COOH terminus of Cbl. This interaction stimulates the ubiquitylation and degradation of the activated EGFR. Furthermore, silencing ITSN by RNA interference attenuated EGFR internalization as well as activation of the extracellular signal-regulated kinasemitogen-activated protein kinase pathway, thereby demonstrating the importance of ITSN in EGFR function. Given the cooperativity between ITSN and additional RTKs, these results point to an important evolutionarily conserved, regulatory role for ITSN in RTK function that is necessary for both signaling from receptors as well as the ultimate termination of receptor signaling.
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