A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Induced by Oxidative Stress Activates Innate Immunity

Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress.Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M(77), thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes.A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure

Quantification of ligand binding to g-protein coupled receptors on cell membranes by ellipsometry

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins

Between- and within-subject covariance perspectives matter for investigations into the relationship between single- and dual-tasking performance

We expand the usually cross-sectional perspective on dual-tasking performance to include both intra- and interpersonal variability, which should capture within-person dynamics and psychological processes better. Two simple tasks, first as single-, then as dual-tasks, were performed by 58 participants over 20 sessions. We found positive relationships between (1) single- and dual-tasking performance and (2) the dual-task component tasks both inter- and intrapersonally. Better single-taskers were better dual-taskers and better single-tasking sessions were better dual-tasking sessions. This implies shared capacities that covary both inter- and intraindividually. We conclude that taking intra- and interpersonal variability into account is a promising future perspective

LH-LR and FSH binding to Ishikawa cells analysed by TIRE.

<p>Increase in layer thickness Δd after addition of 100 ng/ml human luteinizing hormone releasing hormone (LH-LR) is shown and compared to binding of 100 ng/ml follicle stimulating hormone (FSH) to Ishikawa cells. n = 3. Standards errors are shown.</p

Inhibition by AMD3100 of CXCL12α binding to Ishikawa cells analysed by TIRE.

<p>Ishikawa cells were incubated with various concentrations of the inhibitor AMD3100 as indicated, followed by incubation with the ligand CXCL12α. Significant differences in binding are indicated as follows (n = 3): *** = p≤0.001; ** = p≤0.01. Standards errors are shown.</p

Langmuir-Schaefer deposition of human cells.

<p>Cell droplets were spread over the water surface in the Langmuir trough, and the trough barriers were moved towards each other to compress the cells; the surface pressure diagrams are shown on the right hand side. At a surface pressure Π of 20 mN/m - just before pressure saturation when cells are present in a uniform layer - Cr/Au coated glass slides carrying a positive charge were brought in to contact with the cells on the water surface and removed again, transferring a portion of the cell layer onto the slide.</p

CXCL12α binding to receptors at the cell membrane of SH-SY5Y cells.

<p>Binding of the chemokine CXCL12α to neuronal cells (SH-SY5Y) was analysed by TIRE. Calibration curves (layer thickness increment Δd vs. increasing chemokine concentrations in ng/ml) show binding to native cell membranes deposited on a gold surface. n = 3. Standards errors are shown.</p

Detection of CXCR4 and CXCR7 mRNA in cell lines.

<p>Reverse transcriptase PCR was performed using primer combinations specific for CXCR4 and CXCR7, respectively, with RNA isolated from Ishikawa (Ish) cells as well as from the neuronal cell line SH-SY5Y (SH). PCR-fragment sizes are indicated in basepairs (bp) on the left.</p