Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

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IntroductionAggregation of the high-affinity IgE receptor (Fc⑀RI) on mast cells mediates the allergic response by the release of inflammatory mediators by degranulation and production of cytokines and arachidonic acid metabolites. 1 Fc⑀RI signals by activating nonreceptor-family proteintyrosine kinases (PTKs) Lyn, Syk, and Btk. On aggregation of Fc⑀RI␣ subunits, the Src family PTK Lyn phosphorylates the associated Fc⑀RI␤ and ␥ subunits on tyrosine residues in the immunoreceptor tyrosinebased activating motif. This leads to the recruitment of Syk to the plasma membrane, which is essential for Fc⑀RI-mediated degranulation by phosphorylating downstream molecules. 2 Similarly, Btk is membranetargeted by phosphatidylinositol-3,4,5-triphosphate to its pleckstrinhomology (PH) domain. Two types of kinase Syk and Btk contribute tyrosine phosphorylation and activation of phospholipase C-␥ (PLC-␥) and calcium mobilization. 1,3 Several adaptors and scaffolds lacking enzymatic and transcriptional domains have been identified in the immune receptormediated signaling pathway. Proximal adaptor proteins, which are the substrates of PTKs, have important roles in early Fc⑀RI signaling. They have multiple motifs and domains that allow their binding to the other molecules. Activated PTKs phosphorylate the substrate adaptor proteins to assemble the cytoplasmic signaling molecules in the juxtamembrane region for effective interaction, presumably in glycolipid enriched microdomains (GEMs). Mice deficient in the adaptor protein linker for activation of T cells (LAT) SLP-76 (SH2-containing leukocyte phosphoprotein of 76 kDa) or Gab2 (Grb2-associated binder-2) were resistant to IgE-mediated passive systemic anaphylaxis. 4-6 Analysis of bone marrow-derived mast cells (BMMCs) from mice revealed their essential roles in Fc⑀RI-mediated degranulation and/or cytokine production.3BP2 was originally isolated as an Abl SH3-binding protein of unknown function. 7 In addition to the proline-rich region that mediates SH3 binding, it has PH and Src homology 2 (SH2) domains. 3BP2 was also identified as one of the Syk kinaseinteracting proteins by a yeast 2-hybrid screen. 8 C-terminal SH2 domain of 3BP2 is required to interact with Syk in yeast. Expression of 3BP2 has been reported in T, B, natural killer (NK), and monocytic cell lines. Transient overexpression of 3BP2 in T cells induces transcriptional activation of the interleukin 2 (IL-2) gene. In NK cells, overexpression of 3BP2 by vaccinia virus enhances cytotoxicity. 9 Prior to these findings, a specific motif recognized by 3BP2-SH2 domain was examined by in vitro degenerated peptide library screening. 10 The optimal sequence for the 3BP2-SH2 domain is Tyr-Glu-Asn (YEN) motif.The present experiments demonstrated the expression of 3BP2 in mast cells and the functional importance of 3BP2-SH2 domain in Fc⑀RI-mediated signal transduction. Overexpression of 3BP2-SH2 domains in the rat basophilic leukemia RBL-2H3 cells resulted in a dramatic suppression of the Fc⑀RI-mediated tyrosine phosphorylation of P...

Adaptor protein 3BP2 was originally isolated as a proteintyrosine kinase c-Abl-Src homology 3 (SH3) 1 domain-binding protein of unknown function (1). 3BP2 was also identified as a Syk kinase-interacting protein by the yeast two-hybrid screening (2). Transient overexpression of 3BP2 resulted in a transcriptional activation of nuclear factor of activated T cell and activator protein 1, which is induced by T-cell receptor aggregation. Ser 225 and Ser 277 of 3BP2 were identified as essential sites for interacting with 14-3-3 to negatively regulate the function of 3BP2 in lymphocytes (3). Moreover, infection of 3BP2 wild type into NK cells by vaccinia virus enhanced cell cytotoxicity. An in vitro binding study suggested that phosphorylation of Tyr 183 of 3BP2 could be associated with Vav and phospholipase C-␥ (4). In mast cells, overexpression of the 3BP2-SH2 domain suppressed high affinity IgE receptor (Fc⑀RI)-mediated tyrosine phosphorylation of phospholipase C-␥, Ca 2ϩ mobilization, and degranulation (5). These findings have demonstrated that 3BP2 plays a critical role in hematopoietic cells.To propagate the immunoreceptor signal, adaptor proteins contribute to protein-protein and protein-lipid interactions through multiple domains and/or specific phosphotyrosine-containing sequences. Tyrosine phosphorylation of 3BP2 was observed in NK cells and mast cells by cross-linking Fc␥R and Fc⑀RI, respectively (4, 5). To elucidate the function of 3BP2, it is necessary to determine the protein-tyrosine kinase (PTK) that phosphorylates 3BP2 and its binding partner to assemble a signaling complex through specific phosphotyrosine-containing motifs in 3BP2.The Src family PTK Lyn is associated with Fc⑀RI␤. Upon aggregation of Fc⑀RI, Lyn is critical for phosphorylating Fc⑀RI␤ and -␥ subunits on Tyr residues within the immunoreceptor tyrosine-based activating motif (ITAM) (6 -8). By analogy with studies on Hck, Lyn is thought to be activated by the disassembly of the closed intramolecular interaction by (i) CD45-mediated dephosphorylation of C-terminal regulatory Tyr residue, (ii) binding to SH3 and SH2 ligands, and (iii) autophosphorylation of Tyr in the activation loop (9). What is the binding ligand of the SH3 and SH2 domains of Lyn in Fc⑀RI signaling pathway? Pull-down experiments using glutathione S-transferase (GST)-Lyn-SH2 fusion protein indicated that there were multiple phosphoproteins interacting with the Lyn-SH2 after the antigen stimulation of RBL-2H3 cells (10). In addition, although the displacement of intramolecular SH3 interaction is not well understood, it seems likely that some aggregation-induced change in an associated molecule provides a higher affinity SH3 ligand that binds to the Lyn-SH3 domain (11). The SH3 domain is directed toward the proline-rich region, but such a ligand has not been identified yet in the Fc⑀RI signaling.We isolated nonreceptor type PTK Syk from porcine spleen (12). Syk is expressed in hematopoietic, epithelial, and endothelial cells (13)(14)(15)(16). When the ITAM of Fc⑀RI␥ subunits is...