Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled. Using a novel monoclonal antibody that robustly stains cell-surface TrIP, we demonstrate that TrIP is lost from the surface of activated T cells in a manner dependent on the strength of signaling through the T cell receptor (TCR) and specific downstream signaling pathways, in particular classical PKC isoforms. TrIP expression returns by 24 hours after stimulation, suggesting that it may play a role in resetting TCR signaling at later time points. We also provide evidence that ADAM family proteases are required for both constitutive and stimulation-induced downregulation of TrIP in T cells. Finally, by expressing truncated forms of TrIP in cells, we identify the region in the extracellular stalk domain of TrIP that is targeted for proteolytic cleavage.

NF-κB/RelA signaling in secretoglobin progenitors mediates plasticity and MMP-induced barrier disruption in house dust mite-induced allergic asthma.

The mechanisms how aeroallergens induce sensitization are incompletely understood. The house dust mite (HDM) Dermatophagoides pteronyssius (Der p) is a ubiquitous aeroallergen that represents a major cause of allergic rhinitis and asthma. Herein, we tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure is a rapid activator of NF-κB/RelA in the Secretoglobin (Scgb1a1+) lineage associated with upregulation of NF-κB/RelA-dependent markers of epithelial plasticity. To determine the effect of epithelial NF-κB signaling, NF-κB was depleted in a tamoxifen (TMX)-inducible Scgb1a1-CreERTM mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted [RelA knockdown (KD)] mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of metalloproteinase (MMP)9 that are RelA-dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity, and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type (WT) mice. Loss of functional barrier was associated with MMP disruption of zona occludens (ZO)-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability that allows allergen penetration, sensitization producing allergic asthma (AA) in vivo. We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.NEW & NOTEWORTHY Allergic asthma from house dust mite (HDM) allergy causes substantial morbidity. This study examines the dynamic changes in small airway epithelial cells in a mouse model of HDM exposure. Our findings indicate that NF-κB/RelA signaling mediates matrix metalloproteinase production, disrupting the epithelial barrier resulting in allergic sensitization. Our findings bring new insight into mechanisms for epithelial cell-state change in the allergen response, creating a potential therapeutic pathway for maintaining barrier function in asthma.

Potential genetic polymorphism of matrix metalloproteinase (MMP)-9 in Iranian migraine patients with Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular protozoan parasite that causes neuroinflammation in the brain and a constant need for peripheral leukocyte migration. Matrix metalloproteinase 9 (MMP-9) can play a major role in this neuroinflammation and be implicated in some neurological disorders, such as migraines. Therefore, the genetic polymorphism evaluation of MMP-9 in migraine patients with T. gondii infection was performed. One hundred fourteen migraine patients and 114 healthy controls were evaluated for the presence of anti-Toxoplasma IgG antibodies. Seventy-two migraine patients and 40 healthy controls were randomly selected for assessment of the MMP 9-1562C/T genetic polymorphism. In the preliminary examination, 61 (53.5%) migraine patients and 43 (37.3%) healthy controls were positive for IgG antibodies, with a significant association between T. gondii seropositivity and migraine (OR = 1.90; 95% CI = 1.21-3.223; P = 0.012). Genetic distribution for the polymorphism was not in Hardy-Weinberg equilibrium in cases but showed no significant variation in control groups (P = 0.03 and P = 0.180, respectively). A significant preponderance of the CT + TT genotype was found in migraine subjects compared to controls (P = 0.042) (OR, 1.77, CI, 1.013-2.229). The homozygote muted allele TT had a higher rate in migraine patients (6.9%). There were significant differences in CT + TT genotype between T. gondii positive and negative migraine patients (P = 0.024), but T allele frequencies had no significant variation (OR 1.7 CI, 1.084-2.44 and 0.42 CI, 0.044-3.97, respectively). In conclusion, the results may provide the first evidence for the involvement of the MMP-9 gene polymorphism in the mechanism of migraine pathology following Toxoplasma infection.

A new MMP-mediated prodomain cleavage mechanism to activate bone morphogenetic proteins from the extracellular matrix.

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-ßs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.

Matrix Metalloproteinases in Dental and Periodontal Tissues and Their Current Inhibitors: Developmental, Degradational and Pathological Aspects.

OBJECTIVES: This review article aims to describe some of the roles of Matrix metalloproteinases (MMPs) in enamel, dentine, dental caries, hybrid layer degradation, pulp and periodontal tissues, throwing light on their current inhibitors. The article addresses the potential of MMPs to serve as biomarkers with diagnostic and therapeutic value. DESIGN: The sections of this review discuss MMPs' involvement in developmental, remodeling, degradational and turnover aspects of dental and periodontal tissues as well as their signals in the pathogenesis, progress of different lesions and wound healing of these tissues. The literature was searched for original research articles, review articles and theses. The literature search was conducted in PubMed and MEDLINE for articles published in the last 20 years. RESULTS: 119 published papers, two textbooks and two doctoral theses were selected for preparing the current review. CONCLUSIONS: MMPs are significant proteases, of evident contribution in dental and periapical tissue development, health and disease processes, with promising potential for use as diagnostic and prognostic disease biomarkers. Continuing understanding of their role in pathogenesis and progress of different dental, periapical and periodontal lesions, as well as in dentine-pulp wound healing could be a keystone to future diagnostic and therapeutic regimens.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage.

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/ß, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell-derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Estrogen-induced FOS-like 1 regulates matrix metalloproteinase expression and the motility of human endometrial and decidual stromal cells.

The regulation mechanisms involved in matrix metalloproteinase (MMP) expression and the motility of human endometrial and decidual stromal cells (ESCs and DSCs, respectively) during decidualization remain unclear. DSCs show significant increased cell motility and expression of FOS-like 1 (FOSL1) and MMP1, MMP2, and MMP9 compared with ESCs, whereas lack of decidualization inducers leads to a rapid decrease in FOSL1 and MMP1 and MMP9 expression in DSCs in vitro Therefore, we hypothesized that a link exists between decidualization inducers and FOSL1 in up-regulation of motility during decidualization. Based on the response of ESCs/DSCs to different decidualization systems in vitro, we found that progesterone (P4) alone had no significant effect and that 17ß-estradiol (E2) significantly increased cell motility and FOSL1 and MMP1 and MMP9 expression at the mRNA and protein levels, whereas 8-bromo-cAMP significantly decreased cell motility and FOSL1 and MMP9 expression in the presence of P4. In addition, we showed that E2 triggered phosphorylation of estrogen receptor 1 (ESR1), which could directly bind to the promoter of FOSL1 in ESCs/DSCs. Additionally, we also revealed silencing of ESR1 expression by siRNA abrogated E2-induced FOSL1 expression at the transcript and protein levels. Moreover, silencing of FOSL1 expression by siRNA was able to block E2-induced MMP1 and MMP9 expression and cell motility in ESCs/DSCs. Taken together, our data suggest that, in addition to its enhancement of secretory function, the change in MMP expression and cell motility is another component of the decidualization of ESCs/DSCs, including estrogen-dependent MMP1 and MMP9 expression mediated by E2-ESR1-FOSL1 signaling.

In vitro angiogenesis inhibition with selective compounds targeting the key glycolytic enzyme PFKFB3.

Abnormal glycolytic metabolism contributes to angiogenic sprouting involved in atherogenesis. We investigated the potential anti-angiogenic properties of specific 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) inhibitors in endothelial cells (ECs). ECs were treated with PFKFB3 inhibitors (named PA-1 and PA-2) and their effects on metabolic and functional characteristics of ECs were investigated. The anti-glycolytic compound 3-(pyridinyl)- 1-(4-pyridinyl)- 2-propen-1-one (3PO) was used as reference compound. PFKFB3 expression and activity (IC50 about 3-21 nM) was inhibited upon treatment with both compounds. Glucose uptake and lactate export were measured using commercial assays and showed a partial reduction up to 40%. PFKFB3 inhibition increased intracellular lactate accumulation, and reduced expression of monocarboxylate transporters-1 (MCT1) and MCT4. Furthermore, endothelial cell migration and proliferation assays demonstrated significant reduction upon treatment with both compounds. Matrix- metalloproteinase (MMP) activity, measured by gelatin zymography, and expression was significantly reduced (up to 25%). In addition, PA compounds downregulated the expression of VCAM-1, VE-cadherin, VEGFa, VEGFR2, TGF-ß, and IL-1ß, in inflamed ECs. Finally, PA-1 and PA-2 treatment impaired the formation of angiogenic sprouts measured by both morphogenesis and spheroid-based angiogenesis assays. Our data demonstrate that the anti-glycolytic PA compounds may affect several steps involved in angiogenesis. Targeting the key glycolytic enzyme PFKFB3 might represent an attractive therapeutic strategy to improve the efficacy of cancer treatments, or to be applied in other pathologies where angiogenesis is a detrimental factor.

Structure-Activity Relationships of UTX-121 Derivatives for the Development of Novel Matrix Metalloproteinase-2/9 Inhibitors.

Celecoxib, a nonsteroidal anti-inflammatory drug, has been reported to have antitumor and antimetastatic activities, and it has potential for application in cancer treatments. The expression of matrix metalloproteinase (MMP)-2/9 is strongly correlated with cancer malignancy, and inhibition of these MMPs is believed to be effective in improving the antitumor and antimetastatic effects of drugs. We have previously revealed that UTX-121, which converted the sulfonamide of celecoxib to methyl ester, has more potent MMP-2/9 inhibitory activity than celecoxib. Based on these findings, we identified compounds with improved MMP inhibitory activity through a structure-activity relationship (SAR) study, using UTX-121 as a lead compound. Among them, compounds 9c and 10c, in which the methyl group of the p-tolyl group was substituted for Cl or F, showed significantly higher antitumor activity than UTX-121, and suppressed the expression of MMP-2/9 and activation of pro MMP-2. Our findings suggest that compounds 9c and 10c may be potent lead compounds for the development of more effective antitumor drugs targeting MMP.

Skin-Whitening and Anti-Wrinkle Effects of Bioactive Compounds Isolated from Peanut Shell Using Ultrasound-Assisted Extraction.

Response surface methodology was employed to optimize the ultrasound-assisted extraction (UAE) conditions for simultaneous optimization of dependent variables, including DPPH radical scavenging activity (RSA), tyrosinase activity inhibition (TAI), and collagenase activity inhibition (CAI) of peanut shell extracts. The effects of the main variables including extraction time (5.0~55.0 min, X1), extraction temperature (26.0~94.0 °C, X2), and ethanol concentration (0.0%~99.5%, X3) were optimized. Based on experimental values from each condition, quadratic regression models were derived for the prediction of optimum conditions. The coefficient of determination (R2) of the independent variable was in the range of 0.89~0.96, which demonstrates that the regression model is suitable for the prediction. In predicting optimal UAE conditions based on the superimposing method, extraction time of 31.2 min, extraction temperature of 36.6 °C, and ethanol concentration of 93.2% were identified. Under these conditions, RSA of 74.9%, TAI of 50.6%, and CAI of 86.8% were predicted, showing good agreement with the experimental values. A reverse transcription polymerase chain reaction showed that peanut shell extract decreased mRNA levels of tyrosinase-related protein-1 and matrix metalloproteinase-3 genes in B16-F0 cell. Therefore, we identified the skin-whitening and anti-wrinkle effects of peanut shell extracts at protein as well as gene expression levels, and the results show that peanut shell is an effective cosmetic material for skin-whitening and anti-wrinkle effects. Based on this study, peanut shell, which was considered a byproduct, can be used for the development of healthy foods, medicines, and cosmetics.

Acer tataricum subsp. ginnala Inhibits Skin Photoaging via Regulating MAPK/AP-1, NF-κB, and TGFß/Smad Signaling in UVB-Irradiated Human Dermal Fibroblasts.

Skin, the organ protecting the human body from external factors, maintains structural and tensile strength by containing many collagen fibrils, particularly type I procollagen. However, oxidative stress by ultraviolet (UV) exposure causes skin photoaging by activating collagen degradation and inhibiting collagen synthesis. Acer tataricum subsp. ginnala extract (AGE) is a herbal medicine with anti-inflammatory and anti-oxidative effects, but there is no report on the protective effect against skin photoaging. Therefore, we conducted research concentrating on the anti-photoaging effect of Acer tataricum subsp. ginnala (AG) in UVB (20 mJ/cm2)-irradiated human dermal fibroblasts (HDF). Then, various concentrations (7.5, 15, 30 µg/mL) of AGE were treated in HDF for 24 h following UVB irradiation. After we performed AGE treatment, the matrix metalloproteinase1 (MMP1) expression was downregulated, and the type I procollagen level was recovered. Then, we investigated the mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) and nuclear factor-κB (NF-κB) pathway, which induce collagen breakdown by promoting the MMP1 level and pro-inflammatory cytokines. The results indicated that AGE downregulates the expression of the MAPK/AP-1 pathway, leading to MMP1 reduction. AGE inhibits nuclear translocation of NF-κB and inhibitor of nuclear factor-κB (IκB) degradation. Therefore, it downregulates the expression of MMP1 and pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 increased by UVB. Besides, the TGFß/Smad pathway, which is mainly responsible for the collagen synthesis in the skin, was also analyzed. AGE decreases the expression of Smad7 and increases TGFßRII expression and Smad3 phosphorylation. This means that AGE stimulates the TGFß/Smad pathway that plays a critical role in promoting collagen synthesis. Thus, this study suggests that AGE can be a functional material with anti-photoaging properties.

Inhibition of CD44 induces apoptosis, inflammation, and matrix metalloproteinase expression in tendinopathy.

Apoptosis has emerged as a primary cause of tendinopathy. CD44 signaling pathways exert anti-apoptotic and -inflammatory effects on tumor cells, chondrocytes, and fibroblast-like synoviocytes. The aim of this study was to examine the association among CD44, apoptosis, and inflammation in tendinopathy. Expression of CD44 and apoptotic cell numbers in tendon tissue from patients with long head of biceps (LHB) tendinopathy were determined according to the histological grades of tendinopathy. Primary tenocytes from Achilles tendon of Sprague-Dawley rats 1 week after collagenase injection were cultured with an antagonizing antibody against CD44. Treatment responses were determined by evaluating cell viability and expression of tendon-related proliferation markers, inflammatory mediators, and apoptosis. The expression of CD44 and apoptosis were positively correlated with the severity of tendinopathy in the human LHB tendinopathy. Furthermore, CD44 expression and apoptotic cells were co-stained in tendinopathic tendon. Blocking the CD44 signaling pathways in rat primary tenocytes by OX-50 induced cell apoptosis and the elevated levels of cleaved caspase-3. Furthermore, they had decreased cell viability and expression of collagen type I, type III, tenomodulin, and phosphorylated AKT. In contrast, there were elevated levels of inflammatory mediators, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, cyclooxygenase-2, and phosphorylated NF-κB, as well as matrix metalloproteinase (MMP) family members including MMP-1, -3, -9, and -13 in tenocytes upon OX-50 treatment. This study is the first to demonstrate the association of CD44 and apoptosis in tendinopathy. Our data imply that CD44 may play a role in tendinopathy via regulating apoptosis, inflammation, and extracellular matrix homeostasis.

Hyaluronan synthase 2 (HAS2) overexpression diminishes the procatabolic activity of chondrocytes by a mechanism independent of extracellular hyaluronan.

Osteoarthritis (OA) is a progressive degenerative disease of the joints caused in part by a change in the phenotype of resident chondrocytes within affected joints. This altered phenotype, often termed proinflammatory or procatabolic, features enhanced production of endoproteinases and matrix metallo-proteinases (MMPs) as well as secretion of endogenous inflammatory mediators. Degradation and reduced retention of the proteoglycan aggrecan is an early event in OA. Enhanced turnover of hyaluronan (HA) is closely associated with changes in aggrecan. Here, to determine whether experimentally increased HA production promotes aggrecan retention and generates a positive feedback response, we overexpressed HA synthase-2 (HAS2) in chondrocytes via an inducible adenovirus construct (HA synthase-2 viral overexpression; HAS2-OE). HAS2-OE incrementally increased high-molecular-mass HA >100-fold within the cell-associated and growth medium pools. More importantly, our results indicated that the HAS2-OE expression system inhibits MMP3, MMP13, and other markers of the procatabolic phenotype (such as TNF-stimulated gene 6 protein (TSG6)) and also enhances aggrecan retention. These markers were inhibited in OA-associated chondrocytes and in chondrocytes activated by interleukin-1ß (IL1ß), but also chondrocytes activated by lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), or HA oligosaccharides. However, the enhanced extracellular HA resulting from HAS2-OE did not reduce the procatabolic phenotype of neighboring nontransduced chondrocytes as we had expected. Rather, HA-mediated inhibition of the phenotype occurred only in transduced cells. In addition, high HA biosynthesis rates, especially in transduced procatabolic chondrocytes, resulted in marked changes in chondrocyte dependence on glycolysis versus oxidative phosphorylation for their metabolic energy needs.

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

Farnesoid X receptor represses matrix metalloproteinase 7 expression, revealing this regulatory axis as a promising therapeutic target in colon cancer.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of bile acid-activated transcription factors and an important regulator of cell proliferation, apoptosis, and Wnt signaling. Down-regulated expression of FXR plays an important role in some malignancies such as colon cancer, and in rodent models of intestinal neoplasia, FXR knockout increases the size and number of colon tumors. These previous observations implicate FXR as a tumor suppressor, but the underlying molecular mechanisms are unclear. Employing complementary experimental approaches and using human colon cancer specimens, human and murine colon cancer cell lines, and FXR transgenic mice, here we identified an additional, potentially important role for FXR. We observed an inverse relationship between the expression of FXR and matrix metalloproteinase-7 (MMP7), a collagenase and signaling molecule consistently associated with colon cancer progression. We noted that FXR gene ablation increases MMP7 expression. Consistent with this finding, FXR overexpression and a dominant-negative FXR mutation reduced and augmented, respectively, MMP7 expression. Of note, MMP7 was the only MMP gene family member whose expression was down-regulated after FXR activation. FXR-mediated regulation of MMP7 transcription did not require heterodimerization with the retinoid X receptor (RXR), indicating that FXR represses MMP7 expression independently of RXR. Last, we uncovered that FXR suppresses MMP7 transcription by binding to a negative FXR-responsive element in the 5' MMP7 promoter, an event that inhibited colon cancer cell proliferation and invasion. These findings identify the FXR-MMP7 axis as a potential therapeutic target for managing colon cancer.

Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase.

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical "moonlighting" functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

Chondroprotective effects of 4-methylumbelliferone and hyaluronan synthase-2 overexpression involve changes in chondrocyte energy metabolism.

Hyaluronan is a critical component of articular cartilage and partially helps retain aggrecan within the extracellular matrix of this tissue. During osteoarthritis, hyaluronan and aggrecan loss are an early sign of tissue damage. However, our recent attempts to mimic hyaluronan loss with the hyaluronan inhibitor 4-methylumbelliferone (4MU) did not exacerbate arthritis-like features of in vitro models of arthritis, but surprisingly, caused the reverse (i.e. provided potent chondroprotection). Moreover, the protective effects of 4MU did not depend on its role as a hyaluronan inhibitor. To understand the molecular mechanism in 4MU-mediated chondroprotection, we considered recent studies suggesting that shifts in intracellular UDP-hexose pools promote changes in metabolism. To determine whether such metabolic shifts are associated with the mechanism of 4MU-mediated pro-catabolic inhibition, using molecular and metabolomics approaches, we examined whether bovine and human chondrocytes exhibit changes in the contribution of glycolysis and mitochondrial respiration to ATP production rates as well as in other factors that respond to or might drive these changes. Overexpression of either HA synthase-2 or 4MU effectively reduced dependence on glycolysis in chondrocytes, especially enhancing glycolysis use by interleukin-1ß (IL1ß)-activated chondrocytes. The reduction in glycolysis secondarily enhanced mitochondrial respiration in chondrocytes, which, in turn, rescued phospho-AMP-activated protein kinase (AMPK) levels in the activated chondrocytes. Other glycolysis inhibitors, unrelated to hyaluronan biosynthesis, namely 2-deoxyglucose and dichloroacetate, caused metabolic changes in chondrocytes equivalent to those elicited by 4MU and similarly protected both chondrocytes and cartilage explants. These results suggest that fluxes in UDP-hexoses alter metabolic energy pathways in cartilage.

Directed evolution of the metalloproteinase inhibitor TIMP-1 reveals that its N- and C-terminal domains cooperate in matrix metalloproteinase recognition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultrabinders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with WT TIMP-1, with inhibition constants (Ki ) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems.