David H. Lovett

Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation

Progressive renal interstitial fibrosis and tubular atrophy represent the final injury pathway for all commonly encountered forms of renal disease that lead to end-stage renal failure. It has been recently recognized that myofibroblastic cells are the major contributors to the deposition of interstitial collagens. While there are several potential cellular sources of myofibroblasts, attention has focused on the transformation of the organized tubular epithelium to the myofibroblastic phenotype, a process potently driven both in vitro and in vivo by transforming growth factor-beta1 (TGF-beta1). Integrity of the underlying basal lamina provides cellular signals that maintain the epithelial phenotype, and disruption by discrete proteases could potentially initiate the transformation process. We demonstrate that TGF-beta1 coordinately stimulates the synthesis of a specific matrix metalloproteinase, gelatinase A (MMP-2), and its activator protease, MT1-MMP (MMP-14), and that active gelatinase A is absolutely required for epithelial-mesenchymal transformation induced by TGF-beta1. In addition, purified active gelatinase A alone is sufficient to induce epithelial-mesenchymal transformation in the absence of exogenous TGF-beta1. Gelatinase A may also mediate epithelial-mesenchymal transformation in a paracrine manner through the proteolytic generation of active TGF-beta1 peptide. MT1-MMP and gelatinase A were co-localized to sites of active epithelial-mesenchymal transformation and basal lamina disruption in the rat remnant kidney model of progressive renal fibrosis. These studies indicate that a discrete matrix metalloproteinase, gelatinase A, is capable of inducing the complex genetic rearrangements that characterize renal tubular epithelial-mesenchymal transformation.

Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney.

The development of progressive glomerulosclerosis in the renal ablation model has been ascribed to a number of humoral and hemodynamic events, including the peptide growth factor, transforming growth factor-beta 1 (TGF-beta 1). An important role has also been attributed to angiotensin II (AII), which, in addition to its hemodynamic effects, can stimulate transcription of TGF-beta 1. We postulated that increased glomerular production of AII, resulting from enhanced intrinsic angiotensinogen expression, stimulates local TGF-beta 1 synthesis, activating glomerular matrix protein synthesis, and leads to sclerosis. Using in situ reverse transcription, the glomerular cell sites of alpha-1 (IV) collagen, fibronectin, laminin B1, angiotensinogen, and TGF-beta 1 mRNA synthesis were determined at sequential periods following renal ablation. The early hypertrophic phase was associated with global, but transient, increases in the mRNA for alpha-1 (IV) collagen. No changes were noted for fibronectin, TGF-beta 1, and angiotensinogen mRNAs. At 24 d after ablation, at which time sclerosis is not evident, endothelial cells, particularly in the dilated capillaries at the vascular pole, expressed angiotensinogen and TGF-beta 1 mRNAs, as well as fibronectin and laminin B1 RNA transcripts. By 74 d after ablation angiotensinogen and TGF-beta 1 mRNAs were widely distributed among endothelial and mesangial cells, and were particularly prominent in regions of evolving sclerosis. These same regions were also notable for enhanced expression of matrix protein mRNAs, particularly fibronectin. All receptor blockade inhibited angiotensinogen, TGF-beta 1, fibronectin, and laminin B1 mRNA expression by the endothelium. We conclude that, as a result of hemodynamic changes, injured or activated endothelium synthesizes angiotensinogen, triggering a cascade of TGF-beta 1 and matrix protein gene expression with resultant development of the segmental glomerular sclerotic lesion.

A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.

Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury

Chronic kidney disease (CKD) and failure are problems of increasing importance. Regardless of the primary etiology, CKD is characterized by tubular atrophy, interstitial fibrosis, and glomerulosclerosis. It has been assumed that diminished matrix metalloproteinase (MMP) activity is responsible for the accumulation of the extracellular matrix (ECM) proteins and collagens that typify the fibrotic kidney. Here we demonstrate that transgenic renal proximal tubular epithelial expression of a specific enzyme, MMP‐2, is sufficient to generate the entire spectrum of pathological and functional changes characteristic of human CKD. At the earliest point, MMP‐2 leads to structural alterations in the tubular basement membrane, a process that triggers tubular epithelial‐mesenchymal transition, with resultant tubular atrophy, fibrosis and renal failure. Inhibition of MMP‐2, specifically in the early, prefibrotic stages of disease may offer an additional approach for treatment of these disabling disorders.—Cheng, S., Pollock, A. S., Mahimkar, R., Olson, J. L., Lovett, D. H. Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB J. 20, E1248–E1256 (2006)

Glomerular Mesangial Cell-specific Transactivation of Matrix Metalloproteinase 2 Transcription Is Mediated by YB-1

Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chronic glomerular disease. We have previously demonstrated that MC activation is directly linked to high level expression of the matrix metalloproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Marti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074–15083), the transcription of which is regulated in a tissue-specific fashion. Recent studies (Harendza, S., Pollock, A., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18786–18796) delineated a strong cis-acting enhancer element, designated MMP-2 RE1, within the 5′-flanking region of the rat MMP-2 gene. Gel shift, DNA footprint, and transcriptional analyses mapped the enhancer element to a unique 40-base pair (bp) sequence located at −1322 to −1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-bp enhancer element with MC nuclear extracts yielded a single protein of 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screening of a human MC cDNA expression library with MMP-2 RE1 exclusively yielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the shifted complex. Examination of the MMP-2 RE1 sequence revealed an incomplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted with recombinant YB-1 on DMS protection footprinting analysis. YB-1 protein preferentially bound the single-stranded components of the 40-bp MMP-2 RE1 and, with increasing concentrations, formed multimeric complexes. Co-transfection of YB-1 in MC increased the enhancer activity within the context of the native MMP-2 promoter, while transfection of non-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to transcriptional suppression. This study indicates that YB-1 is a major, cell type-specific transactivator of MMP-2 transcription by glomerular mesangial cells.

Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy

The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc-dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP-9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability.

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

Background 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. Methodology and Principal Findings 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 M77, 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. Conclusion 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.

Co-operative interactions between NFAT (nuclear factor of activated T cells) c1 and the zinc finger transcription factors Sp1/Sp3 and Egr-1 regulate MT1-MMP (membrane type 1 matrix metalloproteinase) transcription by glomerular mesangial cells

The transition of normally quiescent glomerular MCs (mesangial cells) to a highly proliferative phenotype with characteristics of myofibroblasts is a process commonly observed in inflammatory diseases affecting the renal glomerulus, the ultimate result of which is glomerulosclerosis. Generation of proteolytically active MMP (matrix metalloproteinase)-2 by the membrane-associated membrane type 1 (MT1)-MMP is responsible for the transition of mesangial cells to the myofibroblast phenotype [Turck, Pollock, Lee, Marti and Lovett (1996) J. Biol. Chem. 271, 15074-15083]. In the present study, we show that the expression of MT1-MMP within the context of MCs is mediated by three discrete cis -acting elements: a proximal non-canonical Sp1 site that preferentially binds Sp1; an overlapping Sp1/Egr-1-binding site that preferentially binds Egr-1; and a more distal binding site for the NFAT (nuclear factor of activated T cells) that binds the NFAT c1 isoform present in MC nuclear extracts. Transfection with an NFAT c1 expression plasmid, or activation of calcineurin with a calcium ionophore, yielded major increases in NFAT c1 nuclear DNA-binding activity, MT1-MMP transcription and protein synthesis, which were additive with the lower levels of transactivation provided by the proximal Sp1 and the overlapping Sp1/Egr-1 sites. Specific binding of NFAT c1 to the MT1-MMP promoter was confirmed by chromatin immunoprecipitation studies, while MT1-MMP expression was suppressed by treatment with the calcineurin inhibitor, cyclosporin A. These studies are the first demonstration that a specific NFAT isoform enhances transcription of an MMP (MT1-MMP) that plays a major role in the proteolytic events that are a dominant feature of acute glomerular inflammation. Suppression of MT1-MMP by commonly used calcineurin inhibitors may play a role in the development of renal fibrosis following renal transplantation.

The prodomain of interleukin 1α interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells

Interleukin 1α (IL‐1α), a 33 kDa precursor, is cleaved releasing the 17 kDa carboxyl‐terminal cytokine IL‐1α to which all of the biological properties of IL‐1α have been attributed. We investigated the potential independent properties of the remaining 16 kDa IL‐1α amino‐terminal propiece by expression in human tumor and primary human cell lines. The IL‐1α propiece produced apoptosis in malignant but not normal cell lines. A minimal fragment comprised of amino acids 55–108 was required for apoptosis. Deletion and mutation studies identified an extended nuclear localization sequence required for nuclear localization, induction of apoptosis and concentration of the IL‐1α propiece in interchromatin granule clusters, concentrations of proteins in the RNA splicing and processing pathways. The IL‐1α propiece interacted with five known components of the RNA splicing/processing pathway, suggesting that the mechanism of action may involve changes in RNA splicing or processing. Expression of the IL‐1α propiece caused a shift in the ratio of Bcl‐Xl/Bcl‐Xs toward the apoptotic direction. Our findings indicate that the IL‐1α propiece induces apoptosis in a range of tumor cells and likely operates through a mechanism involving the RNA processing apparatus and the alternate splicing of apoptosis regulatory proteins.—Pollock, A. S., Turck, J., Lovett, D. H. The prodomain of interleukin‐1 α interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells. FASEB J. 17, 203–213 (2003)

The mesangium and glomerulonephritis

SummaryThe mesangium of the glomerular capillary ultrafilter is a specialized pericapillary tissue. In adult mammals its location is limited to the axial portions of the loop, but it extends peripherally to encircle the capillary in the fetal state and in certain glomerular diseases. It contains predominantly intrinsic mesangial cells, which resemble contractile endocytic capillary pericytes, and which are embedded in the extracellular matrix. In addition, the mesangial space normally harbors few resident Ia-antigen bearing, immune-competent cells and rare transient monocyte-macrophages. Due to its unique location between the fenestrated endothelial lining of the capillary lumen and the glomerular basement membrane, constituting the filtration barrier, the mesangium is prone to deposition of potentially noxious plasma constituents and filtration residues, such as phlogogenic foreign proteins and immune complexes. The determinants of the mesangial entry, uptake and removal of such materials are presently incompletely understood but they are thought to include the amount and nature of the deposit, local hemodynamic factors and the ability of the mesangium to degrade or to eliminate the deposited agent. Histopathologic studies of various human and experimental glomerular diseases reveal that increased mesangial cell proliferation and matrix widening may occur either in direct response to deposits or induced by mediators released from inflammatory cells, such as monocyte-macrophages. While the functional damage to the glomerular filter is usually mild when the reaction is limited to the mesangial space, it is more pronounced when the mesangial abnormalities are secondary to subendothelial deposits of the peripheral capillary wall. Recent experimental data indicate that a mesangial inability in removing deposited material may develop in certain chronic glomerular disorders characterized by marked proteinuria, glomerular hypertension and hyperfiltration or accumulation of matrix material. Such states of mesangial dysfunction may play a critical role in the pathogenesis of progressive glomerular sclerosis. It is concluded that better understanding of the pathophysiology of the mesangium would be valuable for designing more effective diagnostic and therapeutic approaches to patients with glomerular disease.ZusammenfassungIm glomerulären Ultrafilter findet sich das Mesangium als ein spezialisiertes perikapilläres Gewebe. Bei ausgewachsenen Säugern ist es auf die axialen Abschnitte der Kapillarschlingen begrenzt, während es im Foetalstadium und bei manchen glomerulären Erkrankungen die Kapillare völlig umgibt. Die anzahlmäßig weit überwiegenden eigentlichen Mesangiumzellen, die in eine extrazelluläre Matrix eingebettet sind, besitzen die Fähigkeit zur Kontraktion und Endozytose und ähneln somit Kapillarperizyten. Zusätzlich sind im Mesangialraum normalerweise einige Ia-Antigen-positive, immunologisch kompetente Zellen angesiedelt und selten finden sich wandernde Monozyten-Makrophagen. Das Mesangium liegt zwischen dem gefensterten Endothel der Kapillare und der glomerulären Basalmembran, der eigentlichen Filtrationsbarriere. Aŭfgrund dieser ganz besonderen Lage ist das Mesangium prädestiniert für die Aufnahme von möglicherweise schädlichen Plasmabestandteilen und Filtrationsrückständen, wie z.B. entzündungsauslösenden Fremdeiweißen und Immunkomplexen. Die Faktoren, die mesangiale Aufnahme und Eliminierung von derartigen Substanzen bestimmen, sind derzeit nicht genau bekannt. Es wird angenommen, daß hierbei neben Menge und Eigenschaften der Ablagerungen auch hämodynamische Faktoren eine Rolle spielen, sowie die Fähigkeit des Mesangium, die abgelagerten Substanzen abzubauen oder zu entfernen. Histopathologische Untersuchungen verschiedener humaner und tierexperimenteller glomerulärer Erkrankungen zeigen, daß die mesangialen Veränderungen gewöhnlich aus Zellproliferation und Zunahme der Matrix bestehen. Sie stellen entweder eine direkte Reaktion auf die abgelagerte Substanz dar, oder sie werden durch Mediatoren induziert, die von Entzündungszellen, wie z.B. Monozyten-Makrophagen, freigesetzt worden sind. Ist eine solche Reaktion auf den Mesangialraum begrenzt, wird die glomeruläre Funktion gewöhnlich nur geringfügig beeinträchtigt. Hingegen findet sich eine stärker ausgeprägte Funktionseinbuße, wenn die mesangialen Schäden durch subendothelial abgelagertes Material in der peripheren Kapillarschlinge ausgelöst worden sind. Neuere experimentelle Ergebnisse weisen daraufhin, daß sich eine mesangiale Insuffizienz hinsichtlich der Elimination von Ablagerungen bei manchen chronischen glomerulären Störungen entwickeln kann, zumal wenn diese durch starke Proteinurie, glomeruläre Blutdruckerhöhung und Hyperfiltration oder Matrixzunahme gekennzeichnet sind. Derartige Zustände von „mesangialer Dysfunktion“ könnten eine entscheidende Rolle in der Pathogenese von fortschreitender glomerulärer Sklerose spielen. Eine Verbesserung unserer Kenntnisse über die Pathophysiologie des Mesangium wäre von Bedeutung für die Entwicklung wirksamer diagnostischer und therapeutischer Maßnahmen bei Patienten mit glomerulären Krankheiten.