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

<i>In silico</i> analysis of promoters of genes upregulated by NTT-MMP-2.

<p>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.</p

Schematic detailing the distinctive processing, localization and activation mechanisms of three known isoforms of MMP-2.

<p><u>Upper Panel</u>: 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. <u>Middle Panel:</u> 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. <u>Lower Panel</u>: 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.</p

The N-terminal truncated MMP-2 and activation of stress-signaling cascades.

<p>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.</p

Selective cellular trafficking of 68

<p> <b>kDa and 65 </b><b>kDa MMP-2 isoforms in model H9C2 cells.</b> 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 (<b>F</b>ull <b>L</b>ength) or <b>N</b>-<b>T</b>erminal <b>T</b>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.</p