Impact of 4‐hydroxynonenal on matrix metalloproteinase‐9 regulation in lipopolysaccharide‐stimulated RAW 264.7 cells

Tissue degradation and leukocyte extravasation suggest proteolytic destruction of the extracellular matrix (ECM) during severe malaria. Matrix metalloproteinases (MMPs) play an established role in ECM turnover, and increased MMP‐9 protein abundance is correlated with malarial infection. The malaria pigment hemozoin (Hz) is a heme detoxification biomineral that is produced during infection and associated with biologically active lipid peroxidation products such as 4‐hydroxynonenal (HNE) adsorbed to its surface. Hz has innate immunomodulatory activity, and many of its effects can be reproduced by exogenously added HNE. Hz phagocytosis enhances MMP‐9 expression in monocytes; thus, this study was designed to examine the ability of HNE to alter MMP‐9 regulation in activated cells of macrophage lineage. Data show that treatment of lipopolysaccharide‐stimulated RAW 264.7 cells with HNE increased MMP‐9 secretion and activity. HNE treatment abolished the cognate tissue inhibitor of metalloproteinase‐1 protein levels, further decreasing MMP‐9 regulation. Phosphorylation of both p38 mitogen‐activated protein kinase (MAPK) and c‐Jun NH2‐terminal kinase was induced by HNE, but only p38 MAPK inhibition lessened MMP‐9 secretion. These results demonstrate the in vitro ability of HNE to cause MMP‐9 dysregulation in an activated cell model. The findings may extend to myriad pathologies associated with lipid peroxidation and elevated MMP‐9 levels leading to tissue damage. Copyright © 2015 John Wiley & Sons, Ltd.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

MMP‐2 and MMP‐9 as prognostic markers for the early detection of urinary bladder cancer

The present study assessed protein and gene expression levels of tissue inhibitor of metalloproteinase‐2 (TIMP‐2), matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 in urine and blood samples of 50 patients with bladder carcinoma. The expression of TIMP‐2, MMP‐2, and MMP‐9 levels with tumor stage and grade was also assessed. Results showed that the expression levels of MMP‐2 and MMP‐9 in both blood and urine were significantly elevated in group 1 when compared with groups 2 and 3 healthy subjects. The discriminatory ability in the diagnosis of bladder carcinoma of MMP‐2 and MMP‐9 expression was confirmed by receiver operating characteristic curve analysis that revealed a sensitivity and specificity of 100%. MMP‐2 and MMP‐9 levels were not correlated with grade or stage of the tumor. With respect to TIMP‐2 blood and urine levels, results showed a significant decrease in gene expression levels in bladder carcinoma group, whereas, TIMP‐2 protein showed a significant increase in bladder carcinoma.     

Increase of tumor necrosis factor‐α in the blood induces early activation of matrix metalloproteinase‐9 in the brain

Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy.     

The ‘Abtropfung Phenomenon’ Revisited: Dermal Nevus Cells from Congenital Nevi Cannot Activate Matrix Metalloproteinase 2 (MMP‐2)

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro‐matrix metalloproteinase (MMP)‐2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP‐2 was not in its active form, we used Western blot to study the expression of members of the MMP‐2 activation pathway (membrane type 1‐MMP and tissue inhibitor of metalloproteinase‐2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol‐12‐myristate‐13‐acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP‐2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.     

The activities of MMP‐9 and total gelatinase respond differently to substrate coating and cyclic mechanical stretching in fibroblasts and myoblasts

The current study was designed to investigate whether the activities of TGC (total gelatinase and collagenase) as well as MMP‐9 (matrix metalloproteinase‐9, gelatinase B) secreted by the cultured fibroblasts and myoblasts were influenced by the specific extracellular substrates and by cyclic mechanical strain. Fibroblasts (Rat 2) and myoblasts (C2C12) were cultured with either fibronectin, laminin or collagen type I for 24 h and applied with or without a biaxial deformation at 1 Hz using the Flexcell FX‐4000 system. MMP‐9 activity was increased in fibroblasts when the cells were in contact with fibronectin and laminin, while in myoblasts, enhanced activity of the secreted enzyme was only observed when collagen was present. TGC activity expressed from myoblasts was increased in cells growing on all three types of extracellular proteins in response to the mechanical stimulation, but in fibroblasts, such an increase was only observed in cells grown on the laminin coating. In summary, our data demonstrate that the activities of MMP‐9 synthesized by fibroblasts tend to be regulated by the specific extracellular protein the cells are in contact with, whereas the gelatinolytic actions of proteases produced by myoblasts are more responsive to the mechanical deformation.     

Proteomic analysis identifies in vivo candidate matrix metalloproteinase‐9 substrates in the left ventricle post‐myocardial infarction

Matrix metalloproteinase‐9 (MMP‐9) deletion has been shown to improve remodeling of the left ventricle post‐myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP‐9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild‐type (wt) and MMP‐9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2‐DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin‐C, thrombospondin‐1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP‐9 null group, which suggested cleavage by MMP‐9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP‐9. In conclusion, examining infarct tissue from wt and MMP‐9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.     

Matrix metalloproteinase-9,-3 and tissue inhibitor of matrix metalloproteinase-1 in colorectal cancer: relationship to clinicopathological variables

The balance between matrix metalloproteinases (MMPs) and their physiological tissue inhibitors of matrix metalloproteinases (TIMPs) is crucial in tumour invasion and progression. The aim of this study was to investigate the levels of MMP-9, MMP-3 and TIMP-1 in colorectal cancer (CRC) and to evaluate these proteinases and their inhibitor with respect to clinicopathological variables. Activities of pro- and active MMP-9 were measured in paired tumour and distant normal tissue specimens from 43 patients with CRC using gelatin zymography. ELISA was employed for the determination of MMP-9, MMP-3 and TIMP-1 protein expressions. The activity levels of pro- and active MMP-9 and protein expression levels of MMP-9, MMP-3 and TIMP-1 were higher in tumour tissues than in the corresponding normal tissues; the differences being significant for all (p < 0.05), except TIMP-1. Similarly, active MMP-9/proMMP-9 and the ratio of protein expression level of MMP-9-TIMP-1 were found to be significantly higher in tumour tissues ( p < 0.01). Among all the clinicopathological variables investigated, significant correlations were found between MMP-9 and presence of perineural invasion, MMP-3 and lymph node status, TIMP-1 and tumour differentiation, MMP-9/TIMP-1 ratio and histological types ( p < 0.05). In conclusion, MMP-3 was not as notably increased as MMP-9 in tumour tissues. However, different roles may be attributed to MMP-9 and MMP-3 in CRC development and progression. Additionally, assessment of TIMP-1 in relation to MMPs appeared to be crucial in CRC studies to provide a basis for the re-evaluation of the clinical usefulness of TIMP-1 in colorectal cancer.     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell‐matrix and cell‐cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non‐ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase‐3 (MMP‐3) or stromelysin‐1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP‐3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP‐3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP‐3 up‐regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Secretory phospholipase A2 promotes MMP‐9‐mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis

Background information. sPLA₂ (secretory phospholipase A₂) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA₂ during chondrogenesis have not been studied. Results. In the present study, for the first time, we detect the secretion of sPLA₂ during limb development and suggest that sPLA₂ influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA₂ promoted cell death by activating MMP‐9 (matrix metalloproteinase‐9) and increasing type I collagen degradation. The additive chondro‐inhibitory actions were induced by co‐treatment of mp‐BSA (p‐aminophenyl‐mannopyranoside‐BSA), a known ligand of the mannose receptor. Chondro‐inhibitory actions by sPLA₂ were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA₂ (phospholipase A₂) receptor and by inhibition of ERK (extracellular‐signal‐regulated kinase) activity. Conclusions. Taken together, our results suggest that elevated levels of sPLA₂ secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP‐9 in a manner typical of receptor‐mediated signalling and that these events lead to cell death.