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.     

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.     

Proteasome subunit LMP2 is required for matrix metalloproteinase-2 and -9 expression and activities in human invasive extravillous trophoblast cell line

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.     

MMP-14 mediated MMP-9 expression is involved in TGF-beta1-induced keratinocyte migration

The importance of expression of matrix metalloproteinase (MMP) in keratinocyte migration is well established, but its role remains unclear. Here we investigated the function of MMP-14 in TGF-beta1-induced keratinocyte migration. TGF-beta1 stimulated cell migration and the expression of MMP-2, -9 in HaCaT human keratinocyte cells. When we lowered MMP-14 mRNA with siRNA, cell migration, and MMP-9 expression decreased. Furthermore, the MMP-14 siRNA also reduced activation of JNK in response to TGF-beta1, and a JNK-specific inhibitor decreased both cell migration and MMP-9 expression. Taken together, these results suggest that TGF-beta1-induced HaCaT cell migration is mediated by MMP-14, which regulates MMP-9 expression via JNK signaling.     

Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.     

Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: Involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways

Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation.     

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.     

Effect of proinflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma on epithelial barrier function and matrix metalloproteinase-9 in Madin Darby canine kidney cells.

BACKGROUND: Elevated matrix metalloproteinase-9 production during inflammation may be deleterious to epithelial barrier function. Therefore we examined the effect of proinflammatory cytokines on the expression and regulation of matrix metalloproteinase-9 in a model renal epithelial cell system. Tight junctions limit diffusion between compartments and permit directional transport of solutes. Impairment of these junctional complexes by proteolysis may contribute to renal failure through loss of barrier function. METHODS: The renal epithelial cell model, MDCK cells were employed to examine metalloproteinase activity and mRNA expression. Epithelial barrier function was determined using paracellular flux studies. RESULTS: We found that matrix metalloproteinase-9 expression (MMP-9) and activity is markedly elevated in response to tumor necrosis factor-alpha exposure through a mitogen-activated protein kinase dependent pathway. The MMP-9 is predominately secreted into the apical compartment and elevated MMP-9 expression correlates with impaired cell barrier function that was restored using a specific inhibitor of MMP activity. Addition of recombinant MMP-9 to the apical compartment of MDCK cultures significantly elevated paracellular flux rate. CONCLUSIONS: We provide direct evidence for a MMP-9-mediated mechanism that produces junctional disruption. Collectively, these findings support the hypothesis that impaired epithelial barrier function due to activation of tissue/matrix degrading mechanisms occurs in response to specific inflammatory cues.     

Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.     

Matrix metalloproteinase expression correlates with virulence following neurotropic mouse hepatitis virus infection

The relationship(s) between viral virulence and matrix metalloproteinase (MMP) expression in the central nervous system (CNS) of mice undergoing lethal and sublethal infections with neurotropic mouse hepatitis virus was investigated. Lethal infection induced increased levels of MMP-3 and MMP-12 mRNAs as well as that of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) compared to sublethal infection. Increased induction of MMP, TIMP, and chemokine expression correlated with increased virus replication but not with inflammatory cell infiltration. Infection of immunosuppressed mice suggested that expression of most MMP, TIMP, and chemokine mRNA was induced primarily in CNS-resident cells. By contrast, MMP-9 protein activity was associated with the infiltration of neutrophils into the CNS. These data indicate an association between the magnitude of inflammatory gene expression within the CNS and viral virulence.     

RNA interference-mediated silencing of laminin receptor 1 (LR1) suppresses migration and invasion and down-regulates matrix metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells: implication in the pathogenesis of preeclampsia

Shallow trophoblast invasion is a common pathological feature of preeclampsia. The 67 kDa laminin receptor 1 (LR1) is a laminin-binding protein that has been reported to be down-regulated in preeclamptic placentas. The aim of the present study was to determine the functional role of LR1 in the migration and invasion of the trophoblast cell line, JEG3 cells. RNA interference mediated by plasmid expressing LR1 short hairpin RNA (shRNA) was utilized to knockdown LR1 expression in JEG3 cells. We found that the mRNA and protein expression levels of LR1 were significantly reduced in LR1-specific shRNA transfected cells compared with the untransfected and control shRNA transfected cells. The wound healing and Transwell invasion assays demonstrated that LR1 knockdown remarkably suppressed the migration and invasion potential of JEG3 cells. The gelatin zymography assay showed that LR1 knockdown greatly reduced matrix metalloproteinase (MMP)-2 and MMP-9 activities in the culture supernatants. Western blot analysis showed that LR1 shRNA significantly decreased expression levels of MMP-2, MMP-9 and phospho-extracellular signal-regulated kinase, but increased expression levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in comparison to the control vector-transfected cells. In conclusion, our data support an important role for LR1 in regulating trophoblast invasion and migration, and suggest a possible pathological mechanism of preeclampsia.