A novel intracellular role of matrix metalloproteinase-3 during apoptosis of dopaminergic cells

We have previously demonstrated that the active form of matrix metalloproteinase-3 (actMMP-3) is released from dopamine(DA)rgic neurons undergoing apoptosis. Herein, whether actMMP-3 might be generated intracellularly, and if so, whether it is involved in apoptosis of DArgic neurons itself was investigated in primary cultured DArgic neurons of wild-type, MMP-3 knockout animals, and CATH.a cells. During apoptosis, gene expression of MMP-3 is induced, specifically among the various classes of MMPs, generating the proform (55 kDa) which is subsequently cleaved to the catalytically active actMMP-3 (48 kDa) involving a serine protease. Intracellular actMMP-3 activity is directly linked to apoptotic signaling in DArgic cells: (i) Pharmacologic inhibition of enzymatic activity, repression of gene expression by siRNA, and gene deficiency all lead to protection; (ii) pharmacologic inhibition causes attenuation of DNA fragmentation and caspase 3 activation, the indices of apoptosis; and (iii) inhibition of the pro-apoptotic enzyme c- Jun N-terminal protein kinase leads to repression of MMP-3 induction. Under the cell stress condition, MMP-3 is released as actMMP-3 rather than the proform (proMMP-3), and catalytically active MMP-3 added to the medium does not cause cell death. Thus, actMMP-3 seems to have a novel intracellular role in apoptotic DArgic cells and this finding provides an insight into the pathogenesis of Parkinson's disease.     

WAVE3 promotes cell motility and invasion through the regulation of MMP-1, MMP-3, and MMP-9 expression

WAVE3 is a member of the WASP/WAVE family of proteins, which play a critical role in the regulation of actin polymerization, cytoskeleton organization, and cell motility. We show here that knockdown of the WAVE3 protein, using RNA interference in MDA-MB-231 cells, decreases phospho-p38 MAPK levels, but not those of phospho-AKT, phospho-ERK, or phospho-JNK. Knockdown of WAVE3 expression also inhibited the expression levels of MMP-1, MMP-3, and MMP-9, but not MMP-2. MMP production could be restored by PMA treatment, without affecting siRNA-mediated WAVE3 knockdown. The WAVE3-mediated downregulation of p38 activity and MMP production is independent of the presence of both WAVE1 and WAVE2, whose expression levels were not affected by loss of WAVE3. We also show that the downstream effect of the WAVE3 knockdown is the inhibition of cell motility and invasion, coupled with increased actin stress fiber formation, as well as reorganization of focal adhesion complexes. These findings suggest that WAVE3 regulates actin cytoskeleton, cell motility, and invasion through the p38 MAPK pathway and MMP production.     

HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.     

Protection from ataxia-linked apoptosis by gap junction inhibitors

Mutations in the protein kinase C gamma (PKCgamma) gene cause spinocerebellar ataxia type 14 (SCA14), a heterogeneous neurodegenerative disorder. Synthetic peptides (C1B1) serve as gap junction inhibitors through activation of PKCgamma control of gap junctions. We investigated the neuroprotective potential of these peptides against SCA14 mutation-induced cell death using neuronal HT22 cells. The C1B1 synthetic peptides completely restored PKCgamma enzyme activity and subsequent control of gap junctions. PKCgamma SCA14 mutant proteins were shown to cause aggregation which initially resulted in endoplasmic reticulum (ER) stress and cell apoptosis as demonstrated by phosphorylation of PERK on Thr981, activation of caspase-12, increases in BiP/GRP78 protein levels, and consequent activation of caspase-3. Pre-incubation with C1B1 peptides completely abolished these SCA14 effects on ER stress and caspase-3 activation, suggesting that C1B1 peptides protect cells from apoptosis through inhibition of gap junctions by restoration of PKCgamma control of gap junctions, which may result in neuroprotection in SCA14.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.     

Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.     

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.     

Infection with human cytomegalovirus alters the MMP-9/TIMP-1 balance in human macrophages

Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.     

Epithelial and bacterial metalloproteinases and their inhibitors in H. pylori infection of human gastric cells

To test the hypothesis that Helicobacter pylori regulates gastric cell secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), culture media from infected and uninfected human gastric adenocarcinoma (AGS) cells were analyzed by zymography, MMP activity assays, and immunoblotting. AGS cells secreted gelatinolytic (prominently 90 kDa) and caseinolytic (110 kDa) activity together with MMP-1, MMP-3, and TIMP-1, TIMP-2, and TIMP-3 isoforms. H. pylori secreted caseinolytic activity (60 kDa), MMP-3-like enzyme activity, and TIMP-3 immunoreactivity. H. pylori infection increased the 110-kDa caseinolytic activity and induced new gelatinolytic (~35 kDa) and caseinolytic (22 kDa) activities. Infection also increased both basal secretion and activation of MMP-1 and MMP-3, enhanced TIMP-3 secretion, and increased the formation of MMP-3/TIMP-3 complexes. TIMP-1 and TIMP-2 secretion were unchanged. Normal AGS cells showed a pancellular distribution of TIMP-3, with redistribution of immunoreactivity toward sites of bacterial attachment after H. pylori infection. The data indicate that MMP and TIMP secretion by AGS cells is modulated by H. pylori infection and that host MMP-3 and a TIMP-3 homolog expressed by H. pylori mediate at least part of the host cell response to infection.     

Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway

Accumulation of misfolded proteins and alterations in Ca2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1(-/-) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.     

Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells

Variations in the matrix metalloproteinase (MMP)-9 gene are related to the presence and severity of atherosclerosis. The aim of this study was to determine the signaling pathways of MMP-9 in endothelial cells subjected to low fluid shear stress. We found that low fluid shear stress significantly increased MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and phosphorylation of MAPK in cultured human umbilical vein endothelial cells (HUVECs). Inhibition of NF-kappaB resulted in remarkable downregulation of stress-induced MMP-9 expression. Pretreatment of HUVECs with inhibitors of p38 mitogen-activating protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) also led to significant suppression of stress-induced MMP-9 expression and NF-kappaB DNA-binding activity. Similarly, addition of integrins inhibitor to HUVECs suppressed the stress-induced MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and the phosphorylation of p38 MAPK, ERK1/2. Our findings demonstrated that the shear stress-induced MMP-9 expression involved integrins-p38 MAPK or ERK1/2-NF-kappaB signaling pathways.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Expression of gelatinases A and B and their endogenous regulators in immortal and transformed fibroblasts

Matrix metalloproteinases (MMP) play a critical role in tumor invasion and metastasis. The goal of this study was to elucidate peculiarities of expression of gelatinases A and B (MMP-2 and MMP-9), membrane type MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP-2) in immortal (IF) and transformed fibroblasts (TF). The study was carried out using embryo rat fibroblasts, sequentially immortalized with the polyomavirus LT gene and transformed with the E7 gene of human papillomavirus (HPV-16). Papillomaviruses type16 and 18 are the etiological factor for cervical cancer. A primary fibroblast (PF) culture of Fisher rats was used as control. Analysis of TF and IF included determination of MMP-2 and MMP-9 activity by hydrolysis of the specific substrate, radioactive collagen type IV; analysis of MMP spectra by a zymographic assay, and estimation of the mRNA expression by RT-PCR. It was found that: (1) collagenolytic activity of MMP was increased only in TF and it depended on the degree of cell tumorigenicity; (2) the study of MMP spectra revealed the presence of MMP-9 only in TF, whereas MMP-2 was found in IF as well; (3) the mRNA expression of MMP-9, MT1-MMP and TIMP-2 increased in all TF while the MMP-2 expression increased in TF only after TF cell selection on rats; (4) the collagenolytic activity as well as the mRNA expression of MMP-2 and MMP-9 and endogenous regulators (MT1-MMP and TIMP-2) did not change in immortalized fibroblasts compared to the PF culture. The data obtained indicate changes in the ratio enzyme/activator/inhibitor and also suggest a significant increase in the TF destructive potential. MMP-9 is supposed to be a marker of fibroblasts transformed by E7 HPV16 gene in a cell culture.     

Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells

An imbalance in the matrix metalloproteinase (MMP) : tissue inhibitor of MMP (TIMP) ratio may be associated with tissue injury. Here, we studied the regulation of TIMP and MMP gene expression in primary glial cultures to ascertain the factors involved in the regulation of these genes in conditions of inflammatory neuropathology. Astrocytes were found to basally express TIMP-1 and TIMP-3 mRNA while microglia expressed only TIMP-2 mRNA. TIMP-4 mRNA was not detectable in either cell type. Treatment with interferon-alpha (IFN-alpha), IFN-gamma, interleukin-3 (IL-3), IL-6 or tumor necrosis factor-alpha (TNF-alpha) did not alter expression of the TIMP genes. However, in astrocytes, but not in microglia, serum, IL-1beta or lipopolysaccharide (LPS) evoked a dose- and time-dependent increase in TIMP-1 mRNA and a coincident down-regulation of the TIMP-3 gene. Astrocytes were found to express mRNA constitutively for MMPs -3, -11 and -14. In contrast, microglia expressed only MMP-12 mRNA under basal conditions. IL-1beta enhanced MMP-3 mRNA levels while LPS increased the MMP-3, -9, -12, -13 and -14 mRNAs. Our findings reveal that regulatory control of TIMP and MMP gene expression by glial cells is agonist- and cell-type specific, and suggest that innate immune signals govern the temporal and spatial expression patterns of TIMP and MMP genes in neuroinflammatory conditions of the CNS.     

Hydrogen sulfide protects against vascular remodeling from endothelial damage

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 μmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.     

Classical Macrophage Activation Up-Regulates Several Matrix Metalloproteinases through Mitogen Activated Protein Kinases and Nuclear Factor-κB

Remodelling of the extracellular matrix (ECM) and cell surface by matrix metalloproteinases (MMPs) is an important function of monocytes and macrophages. Recent work has emphasised the diverse roles of classically and alternatively activated macrophages but the consequent regulation of MMPs and their inhibitors has not been studied comprehensively. Classical activation of macrophages derived in vitro from un-fractionated CD16(+/-) or negatively-selected CD16(-) macrophages up-regulated MMP-1, -3, -7, -10, -12, -14 and -25 and decreased TIMP-3 steady-state mRNA levels. Bacterial lipopolysaccharide, IL-1 and TNFα were more effective than interferonγ except for the effects on MMP-25, and TIMP-3. By contrast, alternative activation decreased MMP-2, -8 and -19 but increased MMP -11, -12, -25 and TIMP-3 steady-state mRNA levels. Up-regulation of MMPs during classical activation depended on mitogen activated protein kinases, phosphoinositide-3-kinase and inhibitor of κB kinase-2. Effects of interferonγ depended on janus kinase-2. Where investigated, similar effects were seen on protein concentrations and collagenase activity. Moreover, activity of MMP-1 and -10 co-localised with markers of classical activation in human atherosclerotic plaques in vivo. In conclusion, classical macrophage activation selectively up-regulates several MMPs in vitro and in vivo and down-regulates TIMP-3, whereas alternative activation up-regulates a distinct group of MMPs and TIMP-3. The signalling pathways defined here suggest targets for selective modulation of MMP activity.