Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: I. A. Western blot analysis for MMP-2 expression in mitochondrial-enriched fractions from left ventricles of four and twelve month old wild type CD-1 mice (n = 4 for each group). MMP-2 bands with apparent molecular masses of 65 kDa are detected in the mitochondrial fractions from the twelve month old mice, but not in the fractions from four month old mice. (rMMP-2: recombinant full-length 68 kDa MMP-2 protein). I. B. Western blot analysis of mitochondrial-enriched fractions from left ventricles of hypomorphic SR-BI KO/ApoER61 h/h mice fed a normal diet or a high fat atherogenic diet for 30 days (n = 3–4). MMP-2 bands of 65 kDa and 62 kDa are detected in the mitochondrial fractions of mice fed an atherogenic diet. ( shows a mitochondrial fraction run in parallel with recombinant 68 kDa MMP-2). II. In vitro model of transient inhibition of oxidative phosphorylation (OxPhosI). Partial OxPhosI was induced by incubation for 15 minutes in mitochondrial substrate glucose/pyruvate-free DMEM as detailed in Methods, followed by restoration in complete medium. More complete OxPhosI was induced by inclusion of antimycin A (2 µM) and 2-deoxyglucose (10 mM) in substrate-free medium. Westerns blots of mitochondrial-enriched fractions were performed at 24, 48 and 72 hours following OxPhosI. The 65 kDa MMP-2 isoform was detected in the mitochondria-enriched fractions from the H9C2 cells subjected to partial inhibition of OxPhosI and this was increased in the fractions from cells subjected to more complete OxPhosI with antimycin A and 2-deoxyglucose. (rMMP2: recombinant full-length 68 kDa MMP-2).

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Western Blot, Expressing, Recombinant, In Vitro, Inhibition, Incubation

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: I. Schematic diagram of the MMP-2 gene. The full length 68 kDa protein is encoded by a transcript generated by the canonical transcriptional start site (TSS) located in the 5′ flanking region of the MMP-2 gene. M 1 is located within the first exon. Mapped alternate TSS’s are located in the 3′ end of the first intron and are induced by hypoxia or epigenetic stress (arrows). Transcripts generated from these TSS encode a 65 kDa MMP-2 protein beginning at M 77 located within the second exon. Boxes below intron I denote chromatin structures characteristic of a poised promoter and histone marks characteristic of promoter (H3K4me3) and enhancer (H3K4me1) elements from the ENCODE project. Solid boxes above the gene sequence denote mapped DNAse hypersensitivity (DHS) sites. II. The N-terminus of the MMP-2 gene contains three in-frame Kozak consensus sequence capable of translational initiation. The canonical Kozak consensus sequence is displayed with the accompanying consensus sequences flanking the human and murine sequences encoding M 1 , M 77 , and M 96 . III. Experimental confirmation of hypoxia-mediated activation of the alternate TSS in the first intron of the MMP-2 gene. Cardiomyoblast H9C2 cells were maintained in 95% or 1% O 2 for 14 hours, followed by Western blot analysis of mitochondria-enriched fractions. The 65 kDa MMP-2 isoform is detected in fractions isolated from H9C2 cells subjected to hypoxia.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Generated, Sequencing, Activation Assay, Western Blot, Isolation

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: I. Amino acid homology of human and mouse MMP-2 N-terminal domains. There is a high degree of amino acid homology within this domain, including conservation of methionine residues at 77 and 96 relative to the first translational start site. The methionine at position 5 in the human sequence is not conserved. M 1 , M 77 , and M 96 are conserved in all genomic MMP-2 sequences extending to Xenopus leavis. Arrows denote MMP-14 (MT1-MMP) activating cleavage site at N 66 /L 67 and MMP-2 autocatalytic cleavage site at K 79 /F 78 . II. Predicted structure of N-terminal truncated MMP-2: Overall and detailed view of human MMP-2 structure (NCBI PDB code 1CK7). Yellow: N-terminal region deleted in the NTT-MMP-2 protein; magenta: remnant of propeptide present in the NTT-MMP-2 protein; green: catalytic domain; blue: hinge region; pink: hemopexin domain; red spheres: zinc atoms. Deletion of the MMP-2 prodomain exposes the catalytically active zinc and generates the active enzyme.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Sequencing

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: kDa and 65 kDa MMP-2 isoforms in model H9C2 cells. I. Relative distributions of 68 (FL) and 65 kDa MMP-2 (NTT) isoforms in cytosolic and mitochondrial fractions following transient transfection with the respective expression plasmids. The quality of the cytosolic and mitochondrial fractions was assessed by Western blots for KDEL (endoplasmic reticulum), CIV (Complex IV, mitochondrial matrix) and LDH (lactate dehydrogenase, cytosol). A faint band of the 68 kDa MMP-2 isoform is present in the cytosolic fraction of cells transfected with 68 kDa MMP2 cDNA, but not in the mitochondria-enriched fraction. The 65 kDa MMP-2 isoform is present in both the cytosolic and mitochondrial fractions of cells transfected with the NTT-MMP-2 cDNA with a ratio of approximately 3∶1. The mitochondrial fractions did include faintly detectable KDEL bands, consistent with the presence of the mitochondria-associated endoplasmic reticulum in the preparation. II. H9C2 cells were transiently transfected with an empty pcDNA3.1 expression plasmid (-) or expression plasmids encoding either the 68 kDa ( F ull L ength) or N - T erminal T runcated 65 kDa MMP-2. Western blot of the extracellular (conditioned medium) fraction revealed the 68 kDa FL, secreted isoform of MMP-2, while the NTT isoform was not detected. The FL 68 kDa isoform was not detected in mitochondria-enriched fractions, while the 65 kDa NTT isoform was present in this fraction.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: I. H9C2 cells were transfected with increasing concentrations of the NTT-MMP-2 expression plasmid, along with luciferase reporter plasmids for NFAT, NF-κB and IRF (interferon response factor). NTT-MMP-2 enhances inflammatory transcriptional signaling in a concentration-dependent manner (*P<0.05). II. Transient OxPhosI induces activation of NFAT and NF-κB signaling: dependence on MMP-2 activity. H9C2 cells were transfected with NFAT and NF-κB luciferase reporter plasmids and subjected to transient OxPhosI as detailed in Materials and Methods, in the presence or absence of the cyclic peptide MMP-2 inhibitor, CTTHWGFTLCGG (25 µM). III. NTT-MMP-2 degrades mitochondria-associated IκB-α. H9C2 cells were subjected to either transient OxPhosI or transfected with the NTT-MMP-2 expression plasmid. Thereafter the mitochondria were isolated, solubilized and Western blots performed for NF-κB inhibitory IκB-α. Degradation peptide fragments denoted with arrow.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Concentration Assay, Activation Assay, Activity Assay, Isolation, Western Blot

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: The frequency of cognate DNA binding motifs for IRF, NFAT and NF-κB present in 2 kb of the 20 transcripts most up-regulated by NTT-MMP-2 and of 20 randomly chosen transcripts not modified by NTT-MMP-2 were determined by database analysis. Horizontal bars depict the mean of each data set. II. Western blot for IRF7 and IRF1 of nuclear extracts prepared from H9C2 cardiomyoblast cells transfected with a control pcDNA3.1 plasmid (CON), cells transfected with the pcDNA3.1 NTT-MMP2 expression plasmid (NTT-MMP2) and an IRF7 and IRF1 positive control nuclear extract prepared from LPS-stimulated macrophage RAW264.7 cells. Transfection of NTT-MMP2 cDNA induces IRF7, but not IRF1, nuclear localization.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Binding Assay, Modification, Western Blot, Transfection, Plasmid Preparation, Expressing, Positive Control

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0034177

Figure Lengend Snippet: Upper Panel : The mRNA transcript for the full length 68 kDa MMP-2 protein is translated and the latent, enzymatically inactive MMP-2 protein is processed through the Golgi and secretory vesicles to the extracellular space. Latent MMP-2 protein is activated by proteolytic cleavage, primarily by MT1-MMP. This removes the inhibitory prodomain, yielding active 62 kDa MMP-2 protein in the extracellular space where the enzyme degrades extracellular matrix components. Middle Panel: The mRNA for the full length 68 kDa protein is translated and a fraction of the synthesized latent MMP-2 protein escapes the secretory pathway and localizes specifically to sarcomeric proteins, including troponin I. Transient redox stress, such as is induced by ischemia reperfusion injury, generates reactive oxygen species and peroxynitrites which open the cysteine switch. This produces active full length MMP-2 which degrades several components of the sarcomeric apparatus, leading to impaired contractility. Lower Panel : Hypoxia and redox stress activate a latent promoter in the first intron of the MMP-2 gene, thereby generating a N-terminal truncated mRNA transcript. The translated 65 kDa MMP-2 isoform lacks the secretory sequence and inhibitory prodomain and is enzymatically active in the cyotosol and mitochondria. The 65 kDa MMP-2 isoform activates NF-κB and NFAT mitochondrial-nuclear stress signaling, which induction of a pro-inflammatory, pro-apoptotic transcriptome.

Article Snippet: The cDNA encoding the full-length human MMP-2 protein was obtained from Origene.

Techniques: Synthesized, Sequencing