Expression profiling of metalloproteinases and tissue inhibitors of metalloproteinases in normal and degenerate human achilles tendon

To profile the messenger RNA (mRNA) expression for the 23 known genes of matrix metalloproteinases (MMPs), 19 genes of ADAMTS, 4 genes of tissue inhibitors of metalloproteinases (TIMPs), and ADAM genes 8, 10, 12, and 17 in normal, painful, and ruptured Achilles tendons. Tendon samples were obtained from cadavers or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. Total RNA was extracted and mRNA expression was analyzed by quantitative real-time reverse transcription–polymerase chain reaction, normalized to 18S ribosomal RNA. In comparing expression of all genes, the normal, painful, and ruptured Achilles tendon groups each had a distinct mRNA expression signature. Three mRNA were not detected and 14 showed no significant difference in expression levels between the groups. Statistically significant (P < 0.05) differences in mRNA expression, when adjusted for age, included lower levels of MMPs 3 and 10 and TIMP-3 and higher levels of ADAM-12 and MMP-23 in painful compared with normal tendons, and lower levels of MMPs 3 and 7 and TIMPs 2, 3, and 4 and higher levels of ADAMs 8 and 12, MMPs 1, 9, 19, and 25, and TIMP-1 in ruptured compared with normal tendons. The distinct mRNA profile of each tendon group suggests differences in extracellular proteolytic activity, which would affect the production and remodeling of the tendon extracellular matrix. Some proteolytic activities are implicated in the maintenance of normal tendon, while chronically painful tendons and ruptured tendons are shown to be distinct groups. These data will provide a foundation for further study of the role and activity of many of these enzymes that underlie the pathologic processes in the tendon

Pressure induced Superconductor-Insulator transition in the spinel compound CuRh2S4

We performed resistivity measurements in CuRh$_{2}$S$_{4}$ under quasi-hydrostatic pressure of up to 8.0 GPa, and found a pressure induced superconductor-insulator (SI) transition. Initially, with increasing pressure, the superconducting transition temperature $T_c$ increases from 4.7 K at ambient pressure to 6.4 K at 4.0 GPa, but decreases at higher pressures. With further compression, superconductivity in CuRh$_{2}$S$_{4}$ disappears abruptly at a critical pressure $P_{\rm SI}$ between 5.0 and 5.6 GPa, when it becomes an insulator.Comment: 4 pages, 4 figure

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

BACKGROUND: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously. PRINCIPAL FINDINGS: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling. CONCLUSION: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development

A functional AT/G polymorphism in the 5'-untranslated region (UTR) of SETDB2 in the IgE locus on human chromosome 13q14

The immunoglobulin E (IgE)-associated locus on human chromosome 13q14 influencing asthma-related traits contains the genes PHF11 and SETDB2. SETDB2 is located in the same linkage disequilibrium region as PHF11 and polymorphisms within SETDB2 have been shown to associate with total serum IgE levels. In this report, we sequenced the 15 exons of SETDB2 and identified a single previously ungenotyped mutation (AT/G, rs386770867) in the 5′-untranslated region of the gene. The polymorphism was found to be significantly associated with serum IgE levels in our asthma cohort (P=0.0012). Electrophoretic mobility shift assays revealed that the transcription factor Ying Yang 1 binds to the AT allele, whereas SRY (Sex determining Region Y) binds to the G allele. Allele-specific transcription analysis (allelotyping) was performed in 35 individuals heterozygous for rs386770867 from a panel of 200 British families ascertained through probands with severe stage 3 asthma. The AT allele was found to be significantly overexpressed in these individuals (P=1.26 × 10(−21)). A dual-luciferase assay with the pGL3 luciferase reporter gene showed that the AT allele significantly affects transcriptional activities. Our results indicate that the IgE-associated AT/G polymorphism (rs386770867) regulates transcription of SETDB2

Exportin 4 mediates a novel nuclear import pathway for Sox family transcription factors

SRY and other Sox-type transcription factors are important developmental regulators with various implications in human disease. In this study, we identified Exp4 (exportin 4) as an interaction partner of Sox2 in mouse embryonic stem cells and neural progenitors. We show that, besides its established function in nuclear export, Exp4 acts as a bona fide nuclear import receptor for Sox2 and SRY. Thus, Exp4 is an example of a nuclear transport receptor carrying distinct cargoes into different directions. In contrast to a published study, we observed that the import activity of Imp-α (importin-a) isoforms toward Sox2 is negligible. Instead, we found that Imp9 and the Imp-β/7 heterodimer mediate nuclear import of Sox2 in parallel to Exp4. Import signals for the three pathways overlap and include conserved residues in the Sox2 high-mobility group (HMG) box domain that are also critical for DNA binding. This suggests that nuclear import of Sox proteins is facilitated by several parallel import pathways

Quantitative Proteomic Analysis of Human Embryonic Stem Cell Differentiation by 8-Plex iTRAQ Labelling

Analysis of gene expression to define molecular mechanisms and pathways involved in human embryonic stem cells (hESCs) proliferation and differentiations has allowed for further deciphering of the self-renewal and pluripotency characteristics of hESC. Proteins associated with hESCs were discovered through isobaric tags for relative and absolute quantification (iTRAQ). Undifferentiated hESCs and hESCs in different stages of spontaneous differentiation by embryoid body (EB) formation were analyzed. Using the iTRAQ approach, we identified 156 differentially expressed proteins involved in cell proliferation, apoptosis, transcription, translation, mRNA processing, and protein synthesis. Proteins involved in nucleic acid binding, protein synthesis, and integrin signaling were downregulated during differentiation, whereas cytoskeleton proteins were upregulated. The present findings added insight to our understanding of the mechanisms involved in hESC proliferation and differentiation

An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling

Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms

Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes

BRCA1 Interacts with Smad3 and Regulates Smad3-Mediated TGF-β Signaling during Oxidative Stress Responses

BRCA1 is a key regulatory protein participating in cell cycle checkpoint and DNA damage repair networks. BRCA1 plays important roles in protecting numerous cellular processes in response to cell damaging signals. Transforming growth factor-beta (TGF-beta) is a potent regulator of growth, apoptosis and invasiveness of tumor cells. TFG-beta activates Smad signaling via its two cell surface receptors, the TbetaRII and ALK5/TbetaRI, leading to Smad-mediated transcriptional regulation.Here, we report an important role of BRCA1 in modulating TGF-beta signaling during oxidative stress responses. Wild-type (WT) BRCA1, but not mutated BRCA1 failed to activate TGF-beta mediated transactivation of the TGF-beta responsive reporter, p3TP-Lux. Further, WT-BRCA1, but not mutated BRCA1 increased the expression of Smad3 protein in a dose-dependent manner, while silencing of WT-BRCA1 by siRNA decreased Smad3 and Smad4 interaction induced by TGF-beta in MCF-7 breast cancer cells. BRCA1 interacted with Smad3 upon TGF-beta1 stimulation in MCF-7 cells and this interaction was mediated via the domain of 298-436aa of BRCA1 and Smad3 domain of 207-426aa. In addition, H(2)O(2) increased the colocalization and the interaction of Smad3 with WT-BRCA1. Interestingly, TGF-beta1 induced Smad3 and Smad4 interaction was increased in the presence of H(2)O(2) in cells expressing WT-BRCA1, while the TGF-beta1 induced interaction between Smad3 and Smad4 was decreased upon H(2)O(2) treatment in a dose-dependent manner in HCC1937 breast cancer cells, deficient for endogenous BRCA1. This interaction between Smad3 and Smad4 was increased in reconstituted HCC1937 cells expressing WT-BRCA1 (HCC1937/BRCA1). Further, loss of BRCA1 resulted in H(2)O(2) induced nuclear export of phosphor-Smad3 protein to the cytoplasm, resulting decreased of Smad3 and Smad4 interaction induced by TGF-beta and in significant decrease in Smad3 and Smad4 transcriptional activities.These results strongly suggest that loss or reduction of BRCA1 alters TGF-beta growth inhibiting activity via Smad3 during oxidative stress responses