ISOLATION AND CHARACTERIZATION OF TISSUE INHIBITORS OF METALLOPROTEINASES (TIMP-1 AND TIMP-2) FROM HUMAN RHEUMATOID SYNOVIAL-FLUID

OSTHUES A, KNAUPER V, OBERHOFF R, REINKE H, Tschesche H. ISOLATION AND CHARACTERIZATION OF TISSUE INHIBITORS OF METALLOPROTEINASES (TIMP-1 AND TIMP-2) FROM HUMAN RHEUMATOID SYNOVIAL-FLUID. FEBS LETTERS. 1992;296(1):16-20.The tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 were purified to apparent homogeneity from human rheumatoid synovial fluid (HRSF). The inhibitors were isolated by dissociation of non-covalent gelatinase/TIMP complexes. TIMP-1 migrated as a single polypeptide with M(r) 28 500 on SDS-PAGE, while the M(r) of TIMP-2 was 21 000. The inhibitory activity was stable under heat and acid pH. N-terminal sequence data were obtained for the first 15 residues of both inhibitors and showed identity to the human fibroblast inhibitors TIMP-1 and TIMP-2. This is the first demonstration that TIMP-1 and TIMP-2 can be directly purified from human rheumatoid synovial fluid. The complex formation between the metalloproteinase inhibitors and leucocyte metalloproteinases was shown by immunoblotting

FRAGMENTATION OF HUMAN POLYMORPHONUCLEAR-LEUKOCYTE COLLAGENASE

KNAUPER V, OSTHUES A, DECLERCK YA, LANGLEY KE, BLASER J, Tschesche H. FRAGMENTATION OF HUMAN POLYMORPHONUCLEAR-LEUKOCYTE COLLAGENASE. BIOCHEMICAL JOURNAL. 1993;291:847-854.Human polymorphonuclear-leucocyte collagenase (M(r) 64000) shows autoproteolytic degradation to two major fragments of M(r) 40000 and M(r) 27000. N-terminal sequence data and investigation of the substrate specificity of the fragments demonstrate that the M(r)-40 000 fragment corresponds to the catalytic domain, whereas the M(r)-27000 fragment shows no enzymic activity. The activity profile of the M(r)-40000 fragment is comparable with the specificity of the intact active collagenase (M(r) 64000), but the ability to cleave collagen was lost. The enzymic activity of this fragment can be inhibited by either tissue inhibitor of metallo-proteinase (TIMP)-1 or recombinant TIMP-2 in a 1:1 molar ratio. The C-terminal part of the enzyme (M(r) 27000), important for the binding reaction with collagen substrates, is involved in collagenolysis

Fragmentation of human polymorphonuclear-leucocyte collagenase.

Human polymorphonuclear-leucocyte collagenase (M(r) 64,000) shows autoproteolytic degradation to two major fragments of M(r) 40,000 and M(r) 27,000. N-terminal sequence data and investigation of the substrate specificity of the fragments demonstrate that the M(r)-40,000 fragment corresponds to the catalytic domain, whereas the M(r0-27,000 fragment shows no enzymic activity. The activity profile of the M(r)-40,000 fragment is comparable with the specificity of the intact active collagenase (M(r) 64,000), but the ability to cleave collagen was lost. The enzymic activity of this fragment can be inhibited by either tissue inhibitor of metalloproteinase (TIMP)-1 or recombinant TIMP-2 in a 1:1 molar ratio. The C-terminal part of the enzyme (M(r) 27,000), important for the binding reaction with collagen substrates, is involved in collagenolysis

SYNTHESIS OF TISSUE INHIBITOR OF METALLOPROTEINASE-1 (TIMP-1) IN HUMAN HEPATOMA-CELLS (HEPG2) - UP-REGULATION BY INTERLEUKIN-6 AND TRANSFORMING GROWTH FACTOR-BETA-1

KORDULA T, GUTTGEMANN I, ROSEJOHN S, et al. SYNTHESIS OF TISSUE INHIBITOR OF METALLOPROTEINASE-1 (TIMP-1) IN HUMAN HEPATOMA-CELLS (HEPG2) - UP-REGULATION BY INTERLEUKIN-6 AND TRANSFORMING GROWTH FACTOR-BETA-1. FEBS LETTERS. 1992;313(2):143-147.Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase-1 (TIMP-1). We show at mRNA and protein levels that TIMP-1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up-regulated by interleukin-6 (IL-6), transforming growth factor beta1 and phorbol 12-myristate 13-acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL-6-stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP-1 mRNA in liver cells

Direct activation of human neutrophil procollagenase by recombinant stromelysin.

Human neutrophil procollagenase was activated by incubation with recombinant active stromelysin. Activation was achieved by cleavage of the Gly78-Phe79 peptide bond at the end of the propeptide domain in a single-step activation mechanism. In addition, accelerated activation was achieved when N-terminally truncated, latent collagenase (with Phe49 as its N-terminal residue) was incubated with recombinant active stromelysin. Determination of the specific activity of recombinant-stromelysin-activated neutrophil collagenase with dinitrophenyl-octapeptide or type I collagen demonstrated the generation of high specific activity. The specific activity of stromelysin-activated enzyme was considerably higher than that of trypsin- or HgCl2-activated collagenase. Thus human neutrophil collagenase is superactivated, like the homologous fibroblast collagenase [Murphy, Cockett, Stephens, Smith and Docherty (1987) Biochem. J. 248, 265-268]. The occurrence of Phe79 at the N-terminus of the neutrophil collagenase seemed to be critical for superactivation, which is in agreement with data published by Suzuki, Enghild, Morodomi, Salvesen and Nagase [(1990) Biochemistry 29, 10261-10270] on fibroblast collagenase

The oxidized linoleic acid metabolite 12,13-DiHOME mediates thermal hyperalgesia during inflammatory pain

Eicosanoids play a crucial role in inflammatory pain. However, there is very little knowledge about the contribution of oxidized linoleic acid metabolites in inflammatory pain and peripheral sensitization. Here, we identify 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), a cytochrome P450-derived linoleic acid metabolite, as crucial mediator of thermal hyperalgesia during inflammatory pain. We found 12,13-DiHOME in increased concentrations in peripheral nervous tissue during acute zymosan- and complete Freund's Adjuvant-induced inflammatory pain. 12,13-DiHOME causes calcium transients in sensory neurons and sensitizes the transient receptor potential vanilloid 1 (TRPV1)-mediated intracellular calcium increases via protein kinase C, subsequently leading to enhanced TRPV1-dependent CGRP-release from sensory neurons. Peripheral injection of 12,13-DiHOME in vivo causes TRPV1-dependent thermal pain hypersensitivity. Finally, application of the soluble epoxide hydrolase (sEH)-inhibitor TPPU reduces 12,13-DiHOME concentrations in nervous tissue and reduces zymosan- and CFA-induced thermal hyperalgesia in vivo. In conclusion, we identify a novel role for the lipid mediator 12,13-DiHOME in mediating thermal hyperalgesia during inflammatory pain and propose a novel mechanism that may explain the antihyperalgesic effects of sEH inhibitors in vivo