Activation of toll-like receptor-9 induces matrix metalloproteinase-9 expression through Akt and tumor necrosis factor-alpha signaling

CpG oligodeoxunucleotide (ODN) plays an important role in immune cell function. The present study examined whether temporal control of toll-like receptor (TLR)-9 by CpG ODN can regulate the expression of matrix metalloproteinase-9 (MMP-9). CpG ODN induced the release of tumor necrosis factor (TNF)-alpha and the expression of TNF receptor (TNFR)-II, but not of TNFR-I, in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. The endosomal acidification inhibitors, chloroquine or bafilomycin A, inhibited CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expression. CpG ODN induced the phosphorylation of Akt, and the inhibition of Akt by LY294002 suppressed CpG ODN-induced TNF-alpha, TNFR-II, and MMP-9 expressions. Moreover, neutralizing TNF-alpha antibody significantly suppressed CpG ODN-induced MMP-9 expression, suggesting the involvement of TNF-alpha. These observations suggest that CpG ODN may play important roles in macrophage activation by regulating the expression of MMP-9 via a TLR-9/Akt/TNF-alpha-dependent signaling pathway.     

Lost in disruption: Role of proteases in glioma invasion and progression

A characteristic feature of malignant glial tumors (gliomas) is their tendency to diffusely infiltrate the nervous system preventing their complete surgical resection. Proteases play a decisive role in this malignant process, either by degradation of brain extracellular matrix (ECM) components, adhesion molecules, or by regulating the activity of growth and chemotactic factors. Secreted matrix metalloproteinases (MMPs) and ADAMTS proteases (ADAMs with thrombospondin motifs) cleave different ECM components like the proteoglycans (lecticans) aggrecan, versican, neurocan and brevican with selective preferences; they are further regulated by endogenous inhibitors and activating metallo- and serine proteases. Cell surface proteases of the ADAM family (A Disintegrin And Metalloproteinase), but also serine proteases regulate the activity of growth factors and chemokines that act as autocrine / paracrine stimulators within gliomas. Thus, proteases play a decisive role for the spread and growth of gliomas and are prominent targets for their therapy.     

Plaque oxysterols induce unbalanced up-regulation of matrix metalloproteinase-9 in macrophagic cells through redox-sensitive signaling pathways: Implications regarding the vulnerability of atherosclerotic lesions

An imbalance in the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) contributes to atherosclerotic plaque destabilization and rupture. Here we determined whether oxysterols accumulating in advanced atherosclerotic lesions play a role in plaque destabilization. In human promonocytic U937 cells, we investigated the effects of an oxysterol mixture of composition similar to that in advanced human carotid plaques on the expression and synthesis of MMP-9 and its endogenous inhibitors TIMP-1 and TIMP-2. A marked increment of MMP-9 gene expression, but not of its inhibitors, was observed by real-time RT-PCR; MMP-9 gelatinolytic activity was also found increased by gel zymography. Consistently, a net increment of MMP-9 protein level was also observed by immunoblotting. Using antioxidants or specific inhibitors or siRNAs, we demonstrated that the oxysterol mixture induces MMP-9 expression through: (i) overproduction of reactive oxygen species, probably by NADPH-oxidase and mitochondria; (ii) up-regulation of mitogen-activated protein kinase signaling pathways via protein kinase C; and (iii) up-regulation of activator protein-1- and nuclear factor-κB-DNA binding. These results suggest, for the first time, that oxysterols accumulating in advanced atherosclerotic lesions significantly contribute to plaque vulnerability by promoting MMP-9/TIMP-1/2 imbalance in phagocytic cells.     

New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface transducers: Outside-in signaling and relationship to tumor progression

This review focuses on matrix metalloproteinases (MMPs)-2 (gelatinase A) and -9 (gelatinase B), both of which are cancer-associated, secreted, zinc-dependent endopeptidases. Gelatinases cleave many different targets (extracellular matrix, cytokines, growth factors, chemokines and cytokine/growth factor receptors) that in turn regulate key signaling pathways in cell growth, migration, invasion, inflammation and angiogenesis. Interactions with cell surface integral membrane proteins (CD44, αVβ/αβ1/αβ2 integrins and Ku protein) can occur through the gelatinases' active site or hemopexin-like C-terminal domain. This review evaluates the recent literature on the non-enzymatic, signal transduction roles of surface-bound gelatinases and their subsequent effects on cell survival, migration and angiogenesis. Gelatinases have long been drug targets. The current status of gelatinase inhibitors as anticancer agents and their failure in the clinic is discussed in light of these new data on the gelatinases' roles as cell surface transducers — data that may lead to the design and development of novel, gelatinase-targeting inhibitors.     

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

Matrix metalloproteinases and their endogenous inhibitors in neuronal physiology of the adult brain

More than 20 matrix metalloproteinases (MMPs) and four of their endogenous tissue inhibitors (TIMPs) act together to control tightly temporally restricted, focal proteolysis of extracellular matrix. In the neurons of the adult brain several components of the TIMP/MMP system are expressed and are responsive to changes in neuronal activity. Furthermore, functional studies, especially involving blocking of MMP activities, along with the identification of MMP substrates in the brain strongly suggest that this enzymatic system plays an important physiological role in adult brain neurons, possibly being pivotal for neuronal plasticity.     

Omega-3 fatty acids and metabolic syndrome: Effects and emerging mechanisms of action

Epidemiological, human, animal, and cell culture studies show that n−3 fatty acids, especially α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), reduce the risk factors of cardiovascular diseases. EPA and DHA, rather than ALA, have been the focus of research on the n−3 fatty acids, probably due to the relatively inefficient conversion of ALA to EPA and DHA in rodents and humans. This review will assess our current understanding of the effects and potential mechanisms of actions of individual n−3 fatty acids on multiple risk factors of metabolic syndrome. Evidence for pharmacological responses and the mechanism of action of each of the n−3 fatty acid trio will be discussed for the major risk factors of metabolic syndrome, especially adiposity, dyslipidemia, insulin resistance and diabetes, hypertension, oxidative stress, and inflammation. Metabolism of n−3 and n−6 fatty acids as well as the interactions of n−3 fatty acids with nutrients, gene expression, and disease states will be addressed to provide a rationale for the use of n−3 fatty acids to reduce the risk factors of metabolic syndrome.