Renin-angiotensin system gene polymorphisms predict the progression to renal insufficiency among Asians with lupus nephritis.

The renin-angiotensin system (RAS) is a strong candidate as a mediator for the development and progression of lupus nephritis (LN). We performed an ethnically stratified analysis of 642 systemic lupus erythematosus (SLE) patients to determine whether various functional RAS gene polymorphisms are associated with SLE renal outcomes. Patients were genotyped for two angiotensin-converting enzyme (ACE) gene polymorphisms: Alu insertion/deletion (I/D) and 23 949 (CT)(2/3), and for two angiotensinogen (Atg) gene polymorphisms: M235T and C-532T. Multivariate analyses demonstrated associations between the ACE I/D, ACE (CT)(2/3) and Atg C-532T functional polymorphisms and LN among Asians. In stratified analyses among LN cases according to high vs low glomerular filtration rate (GFR), associations remained significant for the ACE D (odds ratio (OR) 5.9, P=0.001) and (CT)(2) (OR 6.2, P=0.001) alleles among Asian subjects with low GFR. Lastly, we found allelic dose-dependent associations between the ACE I/D (P=0.003), ACE (CT)(2/3) (P=0.005) and Atg M235T (P=0.04) polymorphisms, and GFR analyzed as a continuous variable among Asians. These findings suggest a significant role for ACE and Atg gene sequence variation and severity of LN among Asians with SLE.

The hematopoietic transcription factor PU.1 represses gelatinase A transcription in glomerular mesangial cells.

The matrix metalloproteinase gelatinase A plays a key role in the evolution of glomerular injury and is a major contributing factor to the development of glomerulosclerosis. Prior studies have focused on a potent cis-acting enhancer element located in the near 5'-flanking region of the rat and human gelatinase A genes (Harendza, S., Pollock, A. S., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18286-18796; Mertens, P. R., Alfonso-Jaume, M. A., Steinmann, K., and Lovett, D. H. (1999) J. Am. Soc. Nephrol. 10, 2480-2487). Given the combinatorial nature of transcriptional regulation, we examined additional regions of the 5'-flanking region of the rat gelatinase A gene to identify further regulatory elements. In this study the identification of a silencing element located between -1903 and -1847 base pairs of the 5'-flanking region of the rat gelatinase A gene is reported. Sequence analysis, electrophoretic mobility studies, and transfection experiments demonstrate that a specific binding sequence for the hematopoietic transcription factor PU.1 is present within the silencing sequence. PU.1 activity is absolutely required for the expression of silencing activity within the context of transfected glomerular mesangial cells. Western blots identify the PU.1 protein within nuclear extracts of mesangial cells, and cotransfection with a PU.1 expression vector directly augments silencing activity. These studies underscore the complex patterns of gelatinase A transcriptional regulation and also strongly suggest that glomerular mesangial cells are ultimately derived from bone marrow cells.

YB-1 regulation of the human and rat gelatinase A genes via similar enhancer elements.

Experimental and clinical studies strongly suggest that gelatinase A plays a central role in the evolution of glomerular injury and sclerosis. The sequences of the 5' flanking regions of the human and rat gelatinase A genes do not share similarities with other members of the matrix metalloproteinase gene family and are regulated in a distinctive manner. The human and rat gelatinase A genes include regions of significant homology (r2 human; RE-1 rat), which have been shown to act as potent cis-activators of transcription. The rat RE-1 sequence interacts specifically with the developmentally regulated transcription factors AP2 and YB-1, resulting in a synergistic activation of gelatinase A transcription. Although the human r2 sequence specifically interacts with AP2 (Mol Cell Biol 10: 6524-6532, 1990), there is no clear evidence for the presence of a canonical YB-1 binding site (Y-box) within this sequence. This study demonstrates, despite the absence of a canonical Y-box sequence in the r2 element, that YB-1 and AP2 specifically interact with r2, yielding synergistic transactivation of the human gelatinase A gene. It is concluded that the r2 element is the conserved functional analog of the RE-1 element, and that interactions of AP2 and YB-1 govern human gelatinase A gene expression.

A synergistic interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent transcription.

The matrix metalloproteinase gelatinase A plays a central role in several critical physiologic processes, including angiogenesis, tumor invasion/metastasis, and chronic inflammation. We demonstrate that high level gelatinase A expression is mediated by a unique interaction of two developmentally regulated transcription factors, AP2 and YB-1, within a discrete 40-base pair enhancer element (RE-1) located in the 5'-flanking region of the gelatinase A gene. Electrophoretic mobility shift assay studies and immunoprecipitation experiments confirmed a direct interaction of AP2 with this binding sequence in the form of AP2.YB-1 heteromeric complexes. Binding of AP2.YB-1 complexes to the RE-1 sequence results in the formation of extended single-stranded DNA regions and may stabilize DNA conformational changes. Overexpression of YB-1 and AP2 proteins by gelatinase A synthesizing hepatoma HepG2 cells induced a synergistic increase in the RE-1-mediated transcription of nearly 160-fold. Thus, the transcription of gelatinase A is subject to a previously unrecognized interplay of double (AP2) and single-stranded (YB-1) DNA binding transcription factors to yield a highly regulated pattern of gene expression.

Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.

The roles of the matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP), in embryologic development in general, and in nephrogenesis in particular, have not been fully elucidated. The activities of these enzymes and their inhibitors may be critical in the extensive extracellular matrix remodeling that accompanies the formation of the full complement of mature nephrons in the developing kidney. The temporal and spatial expression of two critical basal lamina modifying enzymes, the 72 kDa gelatinase A (MMP-2) and the 92 kDa gelatinase B (MMP-9), as well as TIMP-1, -2, and -3 molecules were evaluated in the developing rat kidney. Additionally, transcripts for the recently described membrane-associated matrix metalloproteinase, MT1-MMP (MMP-14), which can act as an activating receptor for MMP-2/TIMP-2 complexes (Strongin et al.[1995] J. Biol. Chem. 270:5331-5338) were localized by in situ hybridization. Our immunohistochemical data demonstrate distinct localization of MMP-2 within immature nephron structures undergoing epithelial differentiation, while MMP-9 localizes only to the invading vascular structures within immature glomeruli. In contrast, by in situ hybridization, MMP-2 transcripts localize to the background undifferentiated mesenchyme and not to those structures undergoing epithelial differentiation. In a pattern similar to the MMP-2 protein, MT1-MMP transcripts were found within developing epithelial structures. Neither MMP-2, MMP-9 nor MT1-MMP were detected in mature nephrons. TIMP-2 and -3 follow a pattern of expression similar to the MMP-2 protein. We conclude that MMP-2 and TIMP play important roles in the remodeling of basal laminae associated with the epithelial structures of the developing kidney, that these enzymes are temporally and spatially regulated, and that the co-localization of MT1-MMP to sites of basement membrane remodeling suggests a potential role for this molecule as a receptor for and/or modulator of MMP-2/TIMP complexes.

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.

Gelatinase A is a glomerular mesangial cell growth and differentiation factor.

The members of the matrix metalloproteinase gene family play critical roles in numerous physiologic events, including cellular migration, tissue remodeling in wound healing and development, as well as in the evolution of the inflammatory process. The 72 kDa gelatinase A (formerly denoted 72 kDa Type IV collagenase) is centrally involved in the inflammatory and sclerotic events common to most forms of chronic glomerular disease. In this article recent studies are summarized which demonstrate that this particular enzyme can directly affect the proliferative and differentiation properties of the intrinsic glomerular mesangial cell, both in vitro and in vivo.

The N-terminal propiece of interleukin 1 alpha is a transforming nuclear oncoprotein.

Interleukin 1 alpha (IL-1 alpha) is a pleiotropic cytokine involved in the immune response, inflammatory processes, and hematopoiesis, and acts as a mitogen for several malignant cell types, including acute leukemia and Kaposi sarcoma cells. These diverse activities have been exclusively attributed to the plasma membrane receptor-binding, 17-kDa C-terminal component (mature IL-1 alpha) that results from proteolytic processing of the 31- to 33-kDa precursor protein. No biologic function has been ascribed to the unusually large, 16-kDa N-terminal propiece formed as a result of proteolytic processing of IL-1 alpha. We report that the IL-1 alpha N-terminal propiece is concentrated by means of a nuclear localization sequence within the nuclei of both transfected and leukemic cell lines. Overexpression of this component in glomerular mesangial cells, a model perivascular myofibroblast cell type capable of IL-1 alpha synthesis and processing, results in malignant transformation to a spindle cell-type tumor. The functionally bipartite nature of the IL-1 alpha precursor represents a unique combination of the C-terminal, classical cytokine and an N-terminal nuclear oncoprotein. These findings suggest that nuclear transport of the IL-1 alpha N-terminal component may represent a critical component in the transformation of IL-1 alpha-producing cells in the bone marrow or the perivascular area to a malignant phenotype.

Pharmacological inhibition of gelatinase B induction and tumor cell invasion.

The 92 kDa matrix metalloproteinase (gelatinase B, MMP-9) plays a major role in the facilitation of tumor metastasis and in inflammatory disorders characterized by excessive matrix protein destruction. MMP-9 is transcriptionally induced in multiple cell types by exposure to the inflammatory mediators bacterial endotoxin, interleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-alpha). CT-2519, (1-(5-isothiocyanatohexyl)-3,7-dimethylxanthine), a synthetic small molecule from an anti-inflammatory compound library, was evaluated for its effect on endotoxin and cytokine-induced MMP-9 synthesis by a monocytic leukemic cell line, THP-1, and a monocyte/macrophage line, RAW 264.7. CT-2519 dose-dependently inhibited endotoxin and cytokine-induced synthesis of MMP-9 by these cells. Furthermore, both MMP-9 secretion and matrix invasion by cells of a human fibrosarcoma cell line, HT-1080, were inhibited by CT-2519 in a dose-dependent manner. Northern blot analyses and studies utilizing MMP-9 promoter constructs indicated that the inhibitory action of CT-2519 occurs at the level of transcriptional suppression. Given the observation that cellular activation by endotoxin, IL-1 and TNF-alpha may be mediated, at least in part, through induction of certain species of phosphatidic acid (PA), the effect of CT-2519 on lipid levels was analyzed. CT-2519 effectively reduced endotoxin-mediated increases in particular cellular lipid levels. Pharmacologic modulation of cytokine-dependent gene products, such as MMP-9, may offer an important therapeutic approach to the treatment of neoplastic and inflammatory disorders.

Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation.

A biologic role for the 72-kDa gelatinase A (matrix metalloproteinase 2; MMP-2), beyond simple extracellular matrix turnover, was evaluated in glomerular mesangial cells. To determine the significance of MMP-2 secretion for the acquisition of the inflammatory phenotype, we reduced the constitutive secretion of MMP-2 by cultured mesangial cells with antisense RNA expressed by an episomally replicating vector or with specific anti-MMP-2 ribozymes expressed by a retroviral transducing vector. The phenotype of the transfected, or retrovirally infected, cells was profoundly altered from the activated state and closely approximated that of quiescent cells in vivo. The prominent differences included a change in the synthesis and organization of the extracellular matrix, loss of activation markers, and a virtually total exit from the cell cycle. Reconstitution with exogenous active, but not latent MMP-2, induced a rapid return to the inflammatory phenotype in vitro. This effect was specific to MMP-2, because the closely related MMP-9 did not reproduce these changes. Furthermore, this pro-inflammatory effect of MMP-2 is dependent upon the active form of the enzyme, which can be produced by an autocatalytic activation process on the mesangial cell plasma membrane. It is concluded that MMP-2 acts directly upon mesangial cells to permit the development of an inflammatory phenotype. Specific inhibition of MMP-2 activity in vivo may represent an alternate means of ameliorating complex inflammatory processes by affecting the phenotype of the synthesizing cells, per se.

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.

Tissue-specific enhancer-promoter interactions regulate high level constitutive expression of matrix metalloproteinase 2 by glomerular mesangial cells.

The 72-kDa gelatinase A (MMP-2) is a central mediator of the response of the intrinsic glomerular mesangial cell to inflammatory stimuli and is regulated in a unique, cell-specific manner. We isolated a 6-kilobase pair genomic fragment of the rat MMP-2 gene and sequenced and characterized 1686-base pair of the 5'-flanking region. Using a series of 5' deletion constructs of the proximal 5'-flanking region, a strong MMP-2 enhancer element was identified. Gel shift and mutational analyses suggest tha the enhancer region represents the binding site for complex transcription factor demonstrating separable DNA-binding and transcriptional activating domains. The presence and activity of the enhancer element was evaluated in several cell types with varying capabilities to synthesize MMP-2 including mesangial cells, glomerular epithelial cells, and the monocytic U937 cell. Although binding activity was present in all cell types studied, enhancer activity was demonstrated only in mesangial and glomerular epithelial cells. Additional transcriptional control resided in a tissue-specific promoter, which supported transcription only in mesangial cells. These results indicate that the final control of mesangial cell-specific synthesis of MMP-2 derives from an interaction between the strong enhancer element and the tissue-specific MMP-2 promoter.

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.

Structure and expression of the human gene for the matrix metalloproteinase matrilysin.

Matrilysin, a member of the matrix metalloproteinase family, is structurally different from the other matrix metalloproteinases by virtue of the absence of a conserved COOH-terminal protein domain. In addition, matrilysin mRNA is regulated in a specific and distinct manner in normal and malignant tissues. Analysis of the genomic structure of the human matrilysin gene revealed that the organization of the first five exons is highly conserved among the different members of the matrix metalloproteinase family, but that matrilysin contains an atypical sixth exon. The promoter region of the matrilysin gene has several features that are conserved among several other matrix metalloproteinase family members, including the presence of TATA, AP-1, and PEA3 elements. Comparison of the expression of the human matrilysin promoter with rat stromelysin promoter/chloramphenicol acetyltransferase constructs in HeLa cells revealed that constructs containing AP-1 and PEA3 elements respond similarly to epidermal growth factor and tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate) induction, but that the addition of upstream stromelysin sequences results in an increased transcriptional activity not observed with upstream matrilysin sequences. The similarities and differences observed between the promoters of matrilysin and the other metalloproteinases may provide insights into the molecular mechanisms that regulate the expression of this family of enzymes as a whole and the factors that distinguish the expression patterns of individual family members.

Transforming growth factor-beta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.

Transforming growth factor-beta 1 (TGF-beta 1) is generally considered to exert positive effects on the accumulation of extracellular matrices. These occur as the net result of enhanced matrix protein synthesis, diminished matrix metalloproteinase (MMP) synthesis, and augmented production of specific inhibitors, including the tissue inhibitor of metalloproteinases (TIMP-1). Given that glomerular TGF-beta 1 synthesis is induced by inflammation, the effects of this cytokine on synthesis of the 72-kd type IV collagenase and TIMP-1 by cultured human mesangial cells were evaluated. Concentrations of TGF-beta 1 of 5 ng/ml and above specifically stimulated the synthesis of the 72-kd type IV collagenase. This effect was independent of the stimulatory effect of TGF-beta 1 on TIMP-1 synthesis, which was maximal in a lower concentration range (0.1 to 1 ng/ml). Most significantly, the net effect at the higher concentrations of TGF-beta 1 was an excess of enzyme over the TIMP-1 inhibitor. Northern blot analysis of TGF-beta 1-stimulated human mesangial cells demonstrated a specific increase in the abundance of the 3.1 kb mRNA transcript encoding the 72-kd type IV collagenase, presumably mediated by a direct stimulation of 72-kd type IV collagenase mRNA transcription observed as early as 3 hours after exposure to TGF-beta 1. These studies were extended to an analysis of the expression of TGF-beta 1 and 72-kd type IV collagenase mRNAs in normal and nephritic rats. In normal animals, basal TGF-beta 1 and 72-kd type IV collagenase mRNA expression was observed in a strictly mesangial distribution. After induction of acute immune complex-mediated glomerulonephritis, there was a major increase in TGF-beta 1 and 72-kd type IV collagenase mRNA expression, which was strictly limited to the expanded, hypercellular mesangial compartment. Enhanced synthesis of the mesangial type IV collagenase in response to TGF-beta 1 released during glomerular inflammatory processes could have an important role in the extensive glomerular matrix remodeling that accompanies these disorders.

Asymmetric origins of the mature glomerular basement membrane.

Renal plasma filtration is a critical physiologic function that depends upon the precise composition and arrangement of the constituent extracellular matrix proteins within the glomerular basement membrane (GBM). The GBM develops during renal embryogenesis by the fusion of discrete basement membranes produced independently by endothelial and visceral epithelial cells, and, possibly from matrix secreted by the mesangial cells. In the mature animal, however, the epithelial cell has generally been accepted as the sole source of all GBM constituent proteins. Although the final structures and distributions of the component proteins have been defined by histochemical techniques, the individual contributions of the three resident glomerular cell types to the maintenance and turnover of the mature GBM remain uncertain. We report the application of a new technique, in situ reverse transcription (ISRT), for the localization of RNA transcripts of nine major GBM protein components within the closely apposed cells of the glomerulus. Using this technique, we demonstrate that in normal adult rat glomeruli the RNA transcripts for heparan sulfate proteoglycan and the laminin-S chain are primarily expressed by visceral epithelial cells, while Type IV alpha-1 and alpha-2 collagen transcripts were restricted to the endothelial cells in a heterogeneous pattern. RNA transcripts for entactin and the laminin-A and -B2 chains were expressed by all three glomerular cell types, while laminin-B1 and fibronectin transcripts were limited to the mesangium. These findings demonstrate that GBM synthesis in the mature animal is not restricted to the epithelial cell and that all intrinsic glomerular cells contribute to the production of GBM protein components. The ISRT technique also provided the additional, and unexpected, finding that appreciable synthetic heterogeneity exists within individual glomerular cell types.

The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece.

The cytokine interleukin 1 alpha (IL-1 alpha) is a critical mediator of the immune and inflammatory responses. A unique determinant of its activity as compared with IL-1 beta may be its association with the plasma membrane. While the biologic activity of "membrane IL-1" has been extensively reported, the mechanism of membrane binding remains unclear. We report that the N terminus of the 31-kDa IL-1 alpha precursor is myristoylated on specific internal lysine residues. Immunoprecipitation of [3H]myristic acid-radiolabeled human monocyte lysates with IgG antibodies to the 31-kDa IL-1 alpha precursor recovered a protein with the physicochemical properties of the IL-1 alpha N-terminal propiece (16 kDa, pI 4.45). Glycyl N-myristoylation of this protein is precluded by the absence of a glycine residue at position 2, suggesting that the propiece is myristoylated on epsilon-amino groups of lysine. To determine which lysine(s) are acylated, a series of synthetic peptides containing all lysines found in the IL-1 alpha N-terminal propiece were used in an in vitro myristoylation assay containing peptide, myristoyl-CoA, and monocyte lysate as enzyme source. Analysis of the reaction products by reverse-phase HPLC and gas-phase sequencing demonstrated the specific myristoylation of Lys-82 and Lys-83, yielding predominantly monoacylated product. A conserved sequence in the IL-1 beta propiece was myristoylated with at least 8-fold less efficiency. Acylation of the IL-1 alpha precursor by a previously unrecognized lysyl epsilon-amino N-myristoyl-transferase activity may facilitate its specific membrane targeting.

Homology cloning of rat 72 kDa type IV collagenase: cytokine and second-messenger inducibility in glomerular mesangial cells.

Glomerular mesangial cells (MC) play a central role in the synthesis and turnover of the glomerular extracellular matrix. Prior studies [Davies, Thomas, Martin and Lovett (1988) Biochem. J. 251, 419-425; Martin, Davies, Thomas and Lovett (1989) Kidney Int. 36, 790-801] have characterized at the protein level a 72 kDa type IV collagenase that is secreted by cultured human and rat MC. While exposure of most cell types to interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) or phorbol ester has little or even an inhibitory effect on 72 kDa type IV collagenase secretion, these factors significantly increased the synthesis of this enzyme by rat MC. Given this divergent pattern of expression, a homology-based PCR cloning strategy using rat MC cDNA templates was employed to define at the molecular level the structure of the mesangial 72 kDa type IV collagenase. The nucleotide sequence within the open reading frame of the rat mesangial 72 kDa type IV collagenase cDNA diverges from the sequence of the human homologue by approx. 9%. The divergence in the 3' untranslated region was much more extensive. Steady-state levels of the 3.1 kb transcript of the 72 kDa type IV collagenase were low or undetectable in resting MC, but were greatly stimulated following incubation with IL-beta, TNF-alpha or phorbol ester. None of these factors induced synthesis by MC of the closely related 92 kDa type IV collagenase. Synthesis by MC of the 72 kDa type IV collagenase was also induced by second-messenger analogues, including 8-bromo-cyclic AMP and forskolin. It is concluded that MC regulate the expression of this enzyme in an unusual, tissue-specific fashion. Cytokine and second-messenger inducibility may contribute to the enhanced expression of the enzyme during glomerular inflammatory disorders.

[Current aspects in the pathogenesis and therapy of glomerulonephritis: significance of glomerular metalloproteinases]. / Neue Aspekte in der Pathogenese und Therapie der Glomerulonephritiden: die Bedeutung glomerulärer Metalloproteinasen.

The extracellular matrix, comprising the glomerular basement membrane and the mesangial matrix, plays a crucial role in glomerular structure and function. The glomerular extracellular matrix is composed of collagens, proteoglycans and glycoproteins. The distorted balance between synthesis and degradation of extracellular matrix proteins is a hallmark of many forms of glomerulonephritis, such as glomerulosclerosis. The degradation of the matrix occurs through the action of a group of extracellularly active metalloproteinases. Within the glomerulus these enzymes are synthesized by the epithelial and the mesangial cells. The molecular structure of the mesangial metalloproteinases, including their in vitro regulation, was analyzed and the in vivo synthesis of these proteinases was documented for cases of idiopathic rapid progressive glomerulonephritis and anti-Thy 1.1 nephritis. The therapeutic change in the activity and expression of the glomerular metalloproteinases, resulting in the restoration of physiologic matrix metabolic balance, opens up a new perspective for the therapy of glomerular inflammatory processes.