Endothelial-cell-stimulating angiogenesis factor (ESAF) activates progelatinase A (72 kDa type IV collagenase), prostromelysin 1 and procollagenase and reactivates their complexes with tissue inhibitors of metalloproteinases: a role for ESAF in non-inflammatory angiogenesis.
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ABSTRACT:
SUBMITTER: McLaughlin B
PROVIDER: S-EPMC1217547 | biostudies-other | 1996 Aug
REPOSITORIES: biostudies-other
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Project description:To identify structures critical for gelatin-binding of 72 kDa type IV collagenase (gelatinase A), fragments of this metalloproteinase have been expressed in Escherichia coli and assayed for their gelatin affinity. Each of the three fibronectin-related type II domains was found to have affinity for gelatin. Fragments containing all three tandem type II domains had significantly stronger affinity than any of the constituent units, indicating that they co-operate to form the high-affinity gelatin-binding site. Competition experiments have also shown that gelatinase A binds more tightly to gelatin than fibronectin and can displace the latter from denatured collagen.
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Project description:Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.
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Project description: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.
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Project description:Testicular peritubular myoid cells, which have properties similar to those of vascular smooth-muscle cells, secrete a variety of metalloproteinases when maintained in culture in a chemically defined medium. The predominant metalloproteinases secreted were identified as latent type IV procollagenases having molecular masses of 72 kDa and 75 kDa, as detected in Western immunoblots with specific antibodies against type IV procollagenase. When peritubular cells were stimulated by dibutyryl cyclic AMP, forskolin or cholera toxin, they secreted increased amounts of type IV procollagenase. However, little if any of the active type IV collagenase, having a lower molecular mass of 66 kDa, could be detected under these conditions. Addition of low concentrations of cytochalasin D to peritubular cells in monoculture resulted in conversion of the latent type IV collagenase into its active form, assessed with antibody-specificity studies and by the appearance of the 66 kDa protein. In contrast, Sertoli cells in culture did not manifest an increased conversion of type IV procollagenase into type IV collagenase in the presence of cytochalasin D, even though cytochalasin D addition invariably resulted in a disruption of the microfilament assembly in each of these gonadal somatic cell populations. When peritubular cells were co-cultured with Sertoli cells, addition of cytochalasin D no longer resulted in formation of increased amounts of the active form of type IV collagenase. Sertoli cells and peritubular cells each secreted a tissue inhibitor of metalloproteinase type 2, detected with a specific antibody in a Western immunoblot to have a molecular mass of 21 kDa. We conclude that cytochalasin D acts on mesenchymal-type peritubular cells, but not on epithelial-type Sertoli cells, to enhance the conversion of latent type IV procollagenase into active type IV collagenase. This conversion of type IV procollagenase into type IV collagenase by peritubular cells was inhibited by factor(s) secreted by Sertoli cells. Interactions between Sertoli cells and peritubular cells are postulated to modulate net proteinase activities in discrete regions of the testis.
| S-EPMC1151548 | biostudies-other
Project description:The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.
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Project description:Leukemia and lymphoma induced by feline leukemia viruses (FeLVs) are the commonest forms of illness in domestic cats. These viruses do not contain oncogenes, and the source of their pathogenic activity is not clearly understood. Mechanisms involving proto-oncogene activation subsequent to proviral integration and/or development of recombinant viruses with enhanced replication properties are thought to play an important role in their disease pathogenesis. In addition, the long terminal repeat (LTR) regions of these viruses have been shown to be important determinants for pathogenicity and tissue specificity, by virtue of their ability to interact with various transcription factors. Previously, we have shown that, in the case of Moloney murine leukemia virus, the U3 region of the LTR independently induces transcriptional activation of specific cellular genes through an LTR-generated RNA transcript (S. Y. Choi and D. V. Faller, J. Biol. Chem. 269:19691-19694, 1994; S.-Y. Choi and D. V. Faller, J. Virol. 69:7054-7060, 1995). In this report, we show that the U3 region of exogenous FeLV LTRs can induce transcription from collagenase IV (matrix metalloproteinase 9) and monocyte chemotactic protein 1 (MCP-1) promoters up to 12-fold. We also show that AP-1 DNA-binding activity and transcriptional activity are strongly induced in cells expressing FeLV LTRs and that LTR-specific RNA transcripts are generated in those cells. Activation of mitogen-activated protein kinase kinases 1 and 2 (MEK1 and -2) by the LTR is an intermediate step in the FeLV LTR-mediated induction of AP-1 activity. These findings thus suggest that the LTRs of FeLVs can independently activate transcription of specific cellular genes. This LTR-mediated cellular gene transactivation may play an important role in tumorigenesis or preleukemic states and may be a generalizable activity of leukemia-inducing retroviruses.
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Project description:NF-?B is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF-?B have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF-?B monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple-resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA-binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full-length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue-specific labeling and comparison with previously-published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF-?B has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF-?B in the cell.
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Project description:Connective-tissue cells in culture produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that this inhibitor was solely responsible for the inhibition of these enzymes within connective tissue. However, other inhibitors have recently been described, including large inhibitor of metalloproteinases (LIMP) present in the culture medium of human foetal lung fibroblasts. Here we show that a large proportion of the inhibitory activity of LIMP consists of 72,000-M(r)-progelatinase bound to TIMP-2, a recently discovered low-M(r) metalloproteinase inhibitor closely related to TIMP. The physiological implications of the secretion of a complex of 72,000-M(r) progelatinase and TIMP-2 are discussed, and the separation of the complex in 6 M-urea is described.
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Project description:By immunoreactivity analysis using monoclonal antibodies, we showed that the C-terminal domain [R415-631; R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected the immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ("tail" portion of TIMP-2 participates in the binding with the C-terminal domain of proMMP-2. We also demonstrated that not only the C-terminal domain but also the N-terminal domain (R1-417) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of each individual domain to TIMP-2, however, was weak enough that either domain could be fully replaced by proMMP-2 through the same binding sites as does proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 complexed with the N-terminal domain of pro-MMP-2 had fully inhibitory activity against the collagenolytic activity of MMP-1. We also demonstrated that either the interstitial collagenase-TIMP-2 complex or the gelatinase B(MMP-9)-TIMP-2 complex was able to form a ternary complex with proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 complex, but the binding seemed to be less stable than the binding with TIMP-2 alone. Even in the presence of a 10-fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9--TIMP-2 complex. These clear differences might be ascribed to some significant conformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex formation with MMP-9.
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Project description:During angiogenesis, new vessels emerge from existing endothelial lined vessels to promote the degradation of the vascular basement membrane and remodel the extracellular matrix (ECM), followed by endothelial cell migration, and proliferation and the new generation of matrix components. Matrix metalloproteinases (MMPs) participate in the disruption, tumor neovascularization, and subsequent metastasis while tissue inhibitors of metalloproteinases (TIMPs) downregulate the activity of these MMPs. Then, the angiogenic response can be directly or indirectly mediated by MMPs through the modulation of the balance between pro- and anti-angiogenic factors. This review analyzes recent knowledge on MMPs and their participation in angiogenesis.
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