Jan-Olof Winberg

Regulation of matrix metalloproteinase activity in health and disease.

The activity of matrix metalloproteinases (MMPs) is regulated at several levels, including enzyme activation, inhibition, complex formation and compartmentalization. Regulation at the transcriptional level is also important, although this is not a subject of the present minireview. Most MMPs are secreted and have their function in the extracellular environment. This is also the case for the membrane‐type MMPs (MT‐MMPs). MMPs are also found inside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is still poorly understood, although recent studies have unraveled some of their functions. The localization, activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycan core proteins and/or their glycosaminoglycan chains, as well as other molecules. Complexes formed between MMPs and various molecules may also include interactions with noncatalytic sites. Such exosites are regions involved in substrate processing, localized outside the active site, and are potential binding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential for understanding various physiological processes and pathogenesis of diseases, as well as for the development of new MMP targeting drugs.

Matrix metalloproteinases in cancer: their value as diagnostic and prognostic markers and therapeutic targets

Biomarkers are used as tools in cancer diagnostics and in treatment stratification. In most cancers, there are increased levels of one or several members of the matrix metalloproteinases (MMPs). This is a family of proteolytic enzymes that are involved in many phases of cancer progression, including angiogenesis, invasiveness, and metastasis. It has therefore been expected that MMPs could serve as both diagnostic and prognostic markers in cancer patients, but despite a huge number of studies, it has been difficult to establish MMPs as cancer biomarkers. In the present paper, we assess some of the challenges associated with MMP research as well as putative reasons for the conflicting data on the value of these enzymes as diagnostic and prognostic markers in cancer patients. We also review the prognostic value of a number of MMPs in patients with lung, colorectal, breast, and prostate cancers. The review also discusses MMPs as potential target molecules for therapeutic agents and new strategies for development of such drugs.

The Role of Reactive Oxygen Species in Integrin and Matrix Metalloproteinase Expression and Function

Cell adhesion and migration is largely dependent on integrin binding to extracellular matrix, and several signalling pathways involved in these processes have been shown to be modified by reactive oxygen species (ROS). In fact, integrin activation is linked to increased ROS production by NADPH-oxidases, 5-lipoxygenase, and release from mitochondria. Cell migration is intimately linked to degradation of the extracellular matrix, and activated matrix metalloproteinases (MMPs) are a prerequisite for cancer cell invasion and metastasis. In this minireview, we focus on the interplay between integrin-mediated ROS production and MMP expression as well as its biological and pathobiological significance.

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity:

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

Matrix metalloproteinases in subjects with type 1 diabetes.

BackgroundNephropathy is serious complication of diabetes. We have previously shown that level of the proteoglycan syndecan-1 in blood is associated with ultrastructural kidney changes in young persons with type 1 diabetes. Dysregulation of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) may contribute to the development of nephropathy. The aim of this study was to investigate if the levels of MMPs in blood samples are potential markers of early nephropathy in type 1 diabetes.MethodsBlood samples were collected from type 1 diabetes patients after 11 years of diabetes (n = 15) and healthy volunteers (n = 12) and stored at ÷80°C until measurement. Levels and activities of serum MMP-2, MMP-9, TIMP-1 and TIMP- 2 were analyzed and compared to those of control individuals using ELISA, SDS-PAGE gelatin zymography, and Western blot analysis.ResultsThe serum levels of both MMP-9 and MMP-2 were significantly higher in subjects with type 1 diabetes, compared to controls (p = 0.016 and p = 0.008 respectively). Western blotting revealed no differences between the two groups in the levels of TIMP-1 or TIMP-2, respectively.ConclusionOur MMP analysis of serum from a limited number of patients with type 1 diabetes suggest that such analysis is potentially useful as markers in studies of people at risk of progression to chronic kidney disease.

Biological and Pathobiological Functions of Gelatinase Dimers and Complexes

The two matrix metalloproteinases, MMP-2 and MMP-9, are known to form various dimer complexes. In the present review, some of these complexes are described along with their biological and pathobiological functions.

In vitro reconstitution of complexes between pro-matrix metalloproteinase-9 and the proteoglycans serglycin and versican

Previously, we have shown that a proportion of the matrix metalloproteinase‐9 (MMP‐9) synthesized by the macrophage cell line THP‐1 binds to a chondroitin sulfate proteoglycan (CSPG) core protein to form a reduction‐sensitive heteromer. It was also shown that the hemopexin‐like (PEX) domain and the fibronectin‐like (FnII) module in the enzyme are involved in heteromer formation. In this paper, we show that reduction‐sensitive and SDS‐stable heteromers may be reconstituted in vitro by mixing proMMP‐9 with either serglycin, versican or CSPGs isolated from various monocytic cell lines. In addition, a strong but SDS‐soluble proMMP‐9·CSPG heteromer was formed. The two macromolecules in the SDS‐stable reduction‐sensitive heteromers were not linked together by disulfide bonds. As for the heteromer isolated from THP‐1 cells, in vitro reconstituted SDS‐stable and SDS‐soluble heteromers showed weaker binding to gelatin than the proMMP‐9 monomer. Furthermore, gelatin inhibited in vitro reconstitution of the heteromers, showing that the FnII module is involved in the complex formation. Tissue inhibitor of metalloproteinase (TIMP)‐1 was not be detected in the proMMP‐9·CSPG complexes. However, the presence of TIMP‐1 inhibited formation of the SDS‐soluble heteromer, but not the SDS‐stable reduction‐sensitive heteromer. This indicates that different regions in the PEX domain are involved formation of these heteromers.

Interaction of Pro-matrix Metalloproteinase-9/Proteoglycan Heteromer with Gelatin and Collagen

Previously we have shown that THP-1 cells synthesize matrix metalloproteinase-9 (MMP-9) where a fraction of the enzyme is strongly linked to a proteoglycan (PG) core protein. In the present work we show that these pro-MMP-9·PG heteromers have different biochemical properties compared with the monomeric form of pro-MMP-9. In these heteromers, the fibronectin II-like domain in the catalytic site of the enzyme is hidden, and the fibronectin II-like-mediated binding to gelatin and collagen is prevented. However, a fraction of the pro-MMP-9·PG heteromers interacted with gelatin and collagen. This interaction was not through the chondroitin sulfate (CS) part of the PG molecule but, rather, through a region in the PG core protein, a new site induced by the interaction of pro-MMP-9 and the PG core protein, or a non-CS glycosaminoglycan part of the PG molecule. The interaction between pro-MMP-9·PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9·PG heteromers and pro-MMP-9 with approximately the same affinity. Removal of CS chains from the PG part of the heteromers did not affect the binding to gelatin and collagen. Although the identity of the PG core protein is not known, this does not have any impact on the described biochemical properties of the heteromer or its pro-MMP-9 component. It is also shown that a small fraction of the PG, which is not a part of the pro-MMP-9·PG heteromer, can bind gelatin. As for the pro-MMP-9·PG heteromers, this was independent of the CS chains. The structure that mediates the binding of free PG to gelatin is different from the corresponding structure in the pro-MMP-9·PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9·PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced

BackgroundCancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers.MethodsCAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining.ResultsExposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.ConclusionsOur data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

Kinetic interpretations of active site topologies and residue exchanges in Drosophila alcohol dehydrogenases

1. A comparison of full and partly sequenced Adhs from various Drosophila species reveal that 127 of their 253-255 positions are identical (50% identity). 2. Fifty-six of the 115 C-terminal amino acids building up the alcohol binding region differ. In spite of the large differences in primary structure of the alcohol binding region in the Adh enzyme in distantly related Drosophila species, the substrate specificity and stereospecificity have been retained. The topology of the alcohol binding region has been largely conserved during evolution. 3. The primary structures of the alcohol dehydrogenases (Adh) in the Sophophora subgenus is distinguished by few amino acid exchanges, and kinetic and activity parameters show that those at positions 14, 82, 192 and 214 are directly or indirectly involved in coenzyme binding. 4. In these non-metallo Adhs, a tyrosine has been tentatively identified as a nucleophilic catalyst of the hydride transfer step. The three tyrosines at positions 63, 152 and 178 are conserved among the Drosophila alcohol dehydrogenases.