Expression Of Extracellular Proteases Research Articles

Genetic Modification of Aurantiochytrium sp. 18W-13a for Enhancement of Proteolytic Activity by Heterologous Expression of Extracellular Proteases.

A marine thraustochytrid, Aurantiochytrium, is a promising organism to produce docosahexaenoic acid (DHA) and squalene. Utilization of inexpensive substances such as proteins in wastes and by-products from the food industry for cultivation is a considerable option to reduce production cost; however, the proteolytic ability of Aurantiochytrium spp. is low compared to taxonomically close Shizochytrium aggregatum. We previously identified extracellular protease (extracellular protease 1, EP1) in S. aggregatum ATCC 28209 from the supernatant of the culture and found that a similar protease gene (EP2) was located downstream of the EP1 gene. In the present study, we created the transformants expressing SaEP1 and/or SaEP2 to enhance the proteolytic ability of Aurantiochytrium sp. 18W-13a strain and cultivated them in the medium containing casein as a test protein substrate. Through SDS-PAGE analysis, we confirmed that casein in the supernatant was more efficiently degraded by the transformants than the wild type, suggesting that the expressed protease(s) were properly expressed and excreted. After 4-day cultivation in the casein medium, the value of optical density at 660nm and the cell number in the culture of the transformant that expressed both SaEP1 and SaEP2 (designated as EP12 strain) showed 1.48- and 1.38-fold higher than those of wild type, respectively. The DHA and squalene yield of the EP12 strain were respectively 158.3 and 0.23mg L-1, and these values were 1.42- and 2.01-fold higher than those of wild type, respectively, suggesting that the EP12 created in the present study is a favorable strain for the cultivation using protein-containing medium.

Production, Optimization and Partial Characterization of Alkaline Protease from Bacillus subtilis spp. subtilis NRRL B-3384 and B-3387

Bacillus subtilis has been a reliable platform for the expression of extracellular proteases for several decades. Although a majority of Bacillus subtilis subspecies express proteases, the amount of secreted enzyme varies depending on the strain and environmental conditions used. Here, two Bacillus subtilis spp. subtilis strains, NRRL B-3384 and NRRL B-3387, from the ARS Culture collection (NRRL), were compared for secreted protease activity. The highest activity was found in strain NRRL B-3384, and proteolysis occurred at temperatures as high as 80°C and across a broad range of pH, with maximum activity at pH 9.0 and 60°C indicating the presence of a thermostable alkaline protease. To our knowledge, this is the first study to evaluate protease production in Bacillus subtilis spp. subtilis strains NRRL B-3384 and B3387 and suggests that NRRL B-3384 may have utility in the production of enzymes for industrial use.

Effects of deletion of siderophore biosynthesis gene in Pseudomonas fragi on quorum sensing and spoilage ability

Siderophores are important factors in the spoilage process of Pseudomonas fragi, considered to be one of the main spoilage bacterium of tuna, and the secretion of siderophores is regulated by quorum sensing (QS). This study aimed to construct a mutant with the deletion of the siderophore synthetase gene of P. fragi (MS-10), and to explore its effects on the growth, QS, and spoilage potential of P. fragi. The results showed that the deletion of the siderophore biosynthesis gene slowed down the growth rate of the strain. The apoptosis rate increased by 27.7 % compared with that of the wild-type strain at 4 °C for 48 h. Biofilm formation, extracellular protease expression, and signal molecule production were all significantly lower in the mutant strain compared with the wild-type strain. The total viable count and the histamine content showed that the tuna sterile fish block inoculated with the wild-type strain exceeded the acceptable standards by 5 days and was completely spoiled by 7 days, whereas the mutant strain exceeded the acceptable standards by 6 days and was completely spoiled by 9 days. The pH, texture, and other indicators showed that the variation range of the mutant strain was lower than that of the wild-type strain. The deletion of the siderophore biosynthesis gene reduced the spoilage ability of P. fragi. Based on the results, the development of novel preservation agents targeting the control of the siderophore biosynthesis gene could be a new idea for the preservation of aquatic products.

Global Protease Activity Profiling Identifies HER2-Driven Proteolysis in Breast Cancer.

Differential expression of extracellular proteases and endogenous protease inhibitors has been associated with distinct molecular subtypes of breast cancer. However, due to the tight post-translational regulation of protease activity, protease expression-level data alone are not sufficient to understand the role of proteases in malignant transformation. Therefore, we hypothesized that global profiles of extracellular protease activity could more completely reflect differences observed at the transcriptional level in breast cancer and that subtype-associated protease activity may be leveraged to identify specific proteases that play a functional role in cancer signaling. Here, we used a global peptide library-based approach to profile the activities of proteases within distinct breast cancer subtypes. Analysis of 3651 total peptide cleavages from a panel of well-characterized breast cancer cell lines demonstrated differences in proteolytic signatures between cell lines. Cell line clustering based on protease cleavages within the peptide library expanded upon the expected classification derived from transcriptional profiling. An isogenic cell line model developed to further interrogate proteolysis in the HER2 subtype revealed a proteolytic signature consistent with activation of TGF-β signaling. Specifically, we determined that a metalloprotease involved in TGF-β signaling, BMP1, was upregulated at both the protein (2-fold, P = 0.001) and activity (P = 0.0599) levels. Inhibition of BMP1 and HER2 suppressed invasion of HER2-expressing cells by 35% (P < 0.0001), compared to 15% (P = 0.0086) observed in cells where only HER2 was inhibited. In summary, through global identification of extracellular proteolysis in breast cancer cell lines, we demonstrate subtype-specific differences in protease activity and elucidate proteolysis associated with HER2-mediated signaling.

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses.

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.

Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa

ObjectiveObjective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs. Methods3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays. ResultsGC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ. SignificanceDespite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.

Tripeptides From Casein Are Signal Molecules to Induce the Expression of the Extracellular Protease MCP-01 Gene in Marine Bacterium Pseudoalteromonas sp. SM9913.

Microbial extracellular proteases play crucial roles in marine protein degradation and nitrogen recycling. Although a large number of marine bacteria are found to produce extracellular proteases, it is still unknown how marine bacteria respond to environmental proteins to activate the expression of genes encoding extracellular proteases. The inducing signal molecule for marine bacterial extracellular proteases has never been identified. In this study, we identified tripeptides as the inducing signal molecules for the extracellular protease MCP-01 of the deep-sea bacterium Pseudoalteromonas sp. SM9913. We found that casein, but not casamino acids, can induce the gene expression and synthesis of MCP-01, suggesting that peptides rather than amino acids derived from casein induce the gene expression and synthesis of MCP-01 in SM9913. Then, casein was hydrolyzed by SM9913 extracellular proteases, and the peptides with inducing effect were isolated and characterized. Finally, four tripeptides, SPP, RYP, RQF and FRQ, were shown to have significant inducing effect on the expression of MCP-01 gene, indicating that they are likely the inducing signal molecules for the expression of protease MCP-01 gene in SM9913. This study sheds light on the induction mechanism for the gene expression and biosynthesis of marine microbial extracellular proteases, which is helpful in better understanding the adaptation of bacteria to deep-sea sedimental environment.

Hyperglycemia-induced mouse trophoblast spreading is mediated by reactive oxygen species.

During embryo implantation, the outer layer of the blastocyst interacts with the endometrium giving rise to the development of the trophoblast cell lineage. The cells in this lineage participate in the penetration of endometrium due to their motility and invasive properties. The mechanisms that regulate the differentiation and invasive ability of these cells are essential for the establishment and maintenance of an efficient exchange between maternal and fetal tissues during pregnancy. In this context, hyperglycemia can induce oxidative stress causing alterations in the placenta. This study evaluated the role of reactive oxygen species (ROS) in the actions of high glucose concentration (HG) on trophoblast spreading and the expression of extracellular proteases in cultured mouse conceptuses. Blastocysts from gestational day 4 (GD4) were cultured until GD7 in HAM-F10 medium and further treated for 48 hr with HG (25 mM glucose) from GD7 to GD9. This treatment induced larger trophoblast outgrowths and increased ROS concentration, which was associated with increased expression levels of urokinase-type plasminogen activator (PLAU), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9). These effects were prevented by treatment with the non-specific antioxidant N-acetylcysteine (NAC) or apocynin, an inhibitor of NADPH oxidase. Our data suggest that the HG-induced trophoblast spreading and the expression of PLAU, PAI-1, and MMP-9 were mediated by the production of ROS via NADPH oxidase activity. Our results shed light on placental alterations in gestational diabetes mellitus.

A peptide immunization approach to counteract a Staphylococcus aureus protease defense against host immunity

Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

Candidate prognostic markers in breast cancer: focus on extracellular proteases and their inhibitors.

The extracellular matrix (ECM) is the complex network of proteins that surrounds cells in multicellular organisms. Due to its diverse nature and composition, the ECM has a multifaceted role in both normal tissue homeostasis and pathophysiology. It provides structural support, segregates tissues from one another, and regulates intercellular communication. Furthermore, the ECM sequesters a wide range of growth factors and cytokines that may be released upon specific and well-coordinated cues. Regulation of the ECM is performed by the extracellular proteases, which are tasked with cleaving and remodeling this intricate and diverse protein matrix. Accordingly, extracellular proteases are differentially expressed in various tissue types and in many diseases such as cancer. In fact, metastatic dissemination of tumor cells requires degradation of extracellular matrices by several families of proteases, including metalloproteinases and serine proteases, among others. Extracellular proteases are emerging as strong candidate cancer biomarkers for aiding and predicting patient outcome. Not surprisingly, inhibition of these protumorigenic enzymes in animal models of metastasis has shown impressive therapeutic effects. As such, many of these proteolytic inhibitors are currently in various phases of clinical investigation. In addition to direct approaches, aberrant expression of extracellular proteases in disease states may also facilitate the selective delivery of other therapeutic or imaging agents. Herein, we outline extracellular proteases that are either bona fide or probable prognostic markers in breast cancer. Furthermore, using existing patient data and multiple robust statistical analyses, we highlight several extracellular proteases and associated inhibitors (eg, uPA, ADAMs, MMPs, TIMPs, RECK) that hold the greatest potential as clinical biomarkers. With the recent advances in high-throughput technology and targeted therapies, the incorporation of extracellular protease status in breast cancer patient management may have a profound effect on improving outcomes in this deadly disease.

The IclR-Family Regulator BapR Controls Biofilm Formation in B. cenocepacia H111

In Burkholderia cenocepacia H111, the large surface protein BapA plays a crucial role in the formation of highly structured communities, known as biofilms. We have recently demonstrated that quorum sensing (QS) is necessary for the maximal expression of bapA. In this study we identify BapR, a protein from the IclR family of transcriptional regulators that, in conjunction with QS, controls biofilm formation by affecting the expression of bapA. We present evidence that, in addition to bapA, BapR influences the expression of extracellular proteases, swimming motility and has a profound impact in the incidence of persister cells, making this regulator an interesting target for persister cells and biofilm eradication.

LPS Counter Regulates RNA Expression of Extracellular Proteases and Their Inhibitors in Murine Macrophages

Besides their evident importance in host defense, macrophages have been shown to play a detrimental role in different pathological conditions, including chronic inflammation, atherosclerosis, and cancer. Regardless of the exact situation, macrophage activation and migration are intimately connected to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene expression regulation implicated in macrophage-dependent matrix degradation and furthermore emphasize the value of qPCR array techniques for the investigation of the complex regulation of the matrix degradome.

Abstract 5313: Real-time video imaging of protease expression in vivo

Abstract Most new drug candidates generated during in vitro screening turn out to be invalid after time-consuming and costly testing in animal models. Therefore, there is an urgent need for development of noninvasive, real-time, sensitive, and cost-effective tools with high throughput for monitoring and early detection of drug efficacy in vivo. Proteases are known as exceptionally critical signaling proteins that are involved in numerous processes such as cancers. Considerable efforts have been made to identify the role of certain proteases in given biological processes and to screen specific molecules that can regulate protease expression. Most experimental methods are based on the use of protease reporters or molecular beacons. Moreover, they are limited to in vitro applications. To date, protease-activatable probes have already emerged as powerful tools for in vivo imaging because of their potential to offer high specificity and lower background. However, current activatable probes strive to image protease activity and effects in real-time. Although major improvements have been made, such as the conjugation of macromolecules to improve in vivo stability, these probes still suffer in sensitivity and specificity. In addition, conventional protease activatable probes typically take about 24 hours to activate fully, impeding real-time imaging and high throughput in in vivo applications. Here, we report a simple methodology to improve real-time imaging of protease activity by in vivo activatable probes by balancing competing needs for stability and sensitivity in applications where there are high target-to-background signal ratios. We demonstrate the first true real-time in vivo video imaging of extracellular protease expression using an ultrafast-acting and extended-use activatable probe. This simple, one-step technique is capable of boosting fluorescent signals upon target protease cleavage as early as 30 minutes from injection in a small animal model and is able to sustain the strong fluorescent signal up to 24 hours. Using this method, we video imaged the expression and inhibition of matrix metalloproteinases (MMPs) in a MMPs-positive SCC-7 tumor-bearing mouse model. The current platform can be universally applied to any target protease of interest with a known peptide substrate and is adaptable to a wide range of real-time imaging applications with high throughputs such as for in vivo drug screening, examinations of the therapeutic efficacy of drugs, and monitoring of disease onset and development in animal models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5313. doi:10.1158/1538-7445.AM2011-5313

Real-Time Video Imaging of Protease Expression In Vivo

We demonstrate the first true real-time in vivo video imaging of extracellular protease expression using an ultrafast-acting and extended-use activatable probe. This simple, one-step technique is capable of boosting fluorescent signals upon target protease cleavage as early as 30 minutes from injection in a small animal model and is able to sustain the strong fluorescent signal up to 24 hours. Using this method, we video imaged the expression and inhibition of matrix metalloproteinases (MMPs) in a tumor-bearing mouse model. The current platform can be universally applied to any target protease of interest with a known peptide substrate and is adaptable to a wide range of real-time imaging applications with high throughputs such as for in vivo drug screening, examinations of the therapeutic efficacy of drugs, and monitoring of disease onset and development in animal models.

Expression of a Serine Protease Gene prC Is Up-Regulated by Oxidative Stress in the Fungus Clonostachys rosea: Implications for Fungal Survival

BackgroundSoil fungi face a variety of environmental stresses such as UV light, high temperature, and heavy metals. Adaptation of gene expression through transcriptional regulation is a key mechanism in fungal response to environmental stress. In Saccharomyces cerevisiae, the transcription factors Msn2/4 induce stress-mediated gene expression by binding to the stress response element. Previous studies have demonstrated that the expression of extracellular proteases is up-regulated in response to heat shock in fungi. However, the physiological significance of regulation of these extracellular proteases by heat shock remains unclear. The nematophagous fungus Clonostachys rosea can secret an extracellular serine protease PrC during the infection of nematodes. Since the promoter of prC has three copies of the stress response element, we investigated the effect of environmental stress on the expression of prC.Methodology/Principal FindingsOur results demonstrated that the expression of prC was up-regulated by oxidants (H2O2 or menadione) and heat shock, most likely through the stress response element. After oxidant treatment or heat shock, the germination of conidia in the wild type strain was significantly higher than that in the prC mutant strain in the presence of nematode cuticle. Interestingly, the addition of nematode cuticle significantly attenuated the production of reactive oxygen species (ROS) induced by oxidants and heat shock in the wild type strain, but not in prC mutant strain. Moreover, low molecule weight (<3 kD) degradation products of nematode cuticle suppressed the inhibitory effect of conidial germination induced by oxidants and heat shock.Conclusions/SignificanceThese results indicate that PrC plays a protective role in oxidative stress in C. rosea. PrC degrades the nematode cuticle to produce degradation products, which in turn offer a protective effect against oxidative stress by scavenging ROS. Our study reveals a novel strategy for fungi to adapt to environmental stress.

Extracellular protease expression in Microsporum gypseum complex, its regulation and keratinolytic potential

Two soil isolates of Microsporum gypseum were studied for the production of extracellular proteases. Both the strains secreted protease on glucose-gelatin medium. The enzyme activity peaked on day 15 at 28 °C. Asparagine repressed protease yield. Sugars caused catabolite repression of protease formation. Protease activities of both the isolates were significantly affected by incubation period, culture media and carbohydrates used. Both the strains grew on the skin bait and caused a gravimetrically measurable loss of the substrate. Despite less pronounced differences in the keratinase levels, great variations occurred in the amount of keratin degraded by two isolates. Keratinase production as well as loss in substrate mass was better in glucose-lacking flasks than those containing the sugar. Although the rate of keratin degradation was independent of enzyme production, statistically positive correlations were recorded between loss in substrate mass: yielded dry mycelial weight and substrate degradation: keratinase levels.

Tumor Necrosis Factor-related Weak Inducer of Apoptosis Augments Matrix Metalloproteinase 9 (MMP-9) Production in Skeletal Muscle through the Activation of Nuclear Factor-κB-inducing Kinase and p38 Mitogen-activated Protein Kinase: A POTENTIAL ROLE OF MMP-9 IN MYOPATHY

Destruction of skeletal muscle extracellular matrix is an important pathological consequence of many diseases involving muscle wasting. However, the underlying mechanisms leading to extracellular matrix breakdown in skeletal muscle tissues remain unknown. Using a microarray approach, we investigated the effect of tumor necrosis factor-related weak inducer of apoptosis (TWEAK), a recently identified muscle-wasting cytokine, on the expression of extracellular proteases in skeletal muscle. Among several other matrix metalloproteinases (MMPs), we found that the expression of MMP-9, a type IV collagenase, was drastically increased in myotubes in response to TWEAK. The level of MMP-9 was also higher in myofibers of TWEAK transgenic mice. TWEAK increased the activation of both classical and alternative nuclear factor-kappaB (NF-kappaB) signaling pathways. Inhibition of NF-kappaB activity blocked the TWEAK-induced production of MMP-9 in myotubes. TWEAK also increased the activation of AP-1, and its inhibition attenuated the TWEAK-induced MMP-9 production. Overexpression of a kinase-dead mutant of NF-kappaB-inducing kinase or IkappaB kinase-beta but not IkappaB kinase-alpha significantly inhibited the TWEAK-induced activation of MMP-9 promoter. The activation of MMP-9 also involved upstream recruitment of TRAF2 and cIAP2 proteins. TWEAK increased the activity of ERK1/2, JNK1, and p38 MAPK. However, the inhibition of only p38 MAPK blocked the TWEAK-induced expression of MMP-9 in myotubes. Furthermore the loss of body and skeletal muscle weights, inflammation, fiber necrosis, and degradation of basement membrane around muscle fibers were significantly attenuated in Mmp9 knock-out mice on chronic administration of TWEAK protein. The study unveils a novel mechanism of skeletal muscle tissue destruction in pathological conditions.

Osteoblasts up-regulate the expression of extracellular proteases following attachment to Poly(β-hydroxybutyrate-co-β-hydroxyvalerate)

MC3T3-E1 cells demonstrate a lag in osteogenic development when seeded onto Poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV), a biomaterial with substantial potential for bone tissue repair. To determine if this was due to the priority of extracellular matrix (ECM) remodelling over other developmental processes, gene expression levels of proteins involved in the production, maintenance and turnover of the ECM were compared between cells grown on PHBV and tissue culture plastic (TCP) 24 h after seeding. When grown on PHBV, MC3T3-E1 cells up-regulated proteins such as the matrix metalloproteinases and down-regulated the expression of proteins such as collagens that are involved in cell–substrate interactions, but in later-stage processes. The results also suggest that proteins such as fibronectin and aggrecan, and particularly osteopontin, may be more suitable candidates for PHBV functionalization for optimal MC3T3-E1 cell growth than proteins like osteonectin, periostin, vitronectin or collagen. This study confirms the importance of understanding the specific response of therapeutically-relevant cells, such as human stem cells, to candidate biomaterial surfaces in order to achieve optimal regenerative therapies.

Identification of Genes Regulated by the cepIR Quorum-Sensing System in Burkholderia cenocepacia by High-Throughput Screening of a Random Promoter Library

The Burkholderia cenocepacia cepIR quorum-sensing system regulates expression of extracellular proteases, chitinase, and genes involved in ornibactin biosynthesis, biofilm formation, and motility. In a genome-wide screen we identified cepIR-regulated genes by screening a random promoter library of B. cenocepacia K56-2 constructed in a luminescence reporter detection plasmid for differential expression in response to N-octanoyl-l-homoserine lactone (OHL). Eighty-nine clones were identified; in 58 of these clones expression was positively regulated by cepIR, and in 31 expression was negatively regulated by cepIR. The expression profiles of the 89 promoter clones were compared in the cepI mutant K56-dI2 in medium supplemented with 30 pM OHL and K56-2 to confirm that the presence of OHL restored expression to wild-type levels. To validate the promoter library observations and to determine the effect of a cepR mutation on expression of selected genes, the mRNA levels of nine genes whose promoters were predicted to be regulated by cepR were quantitated by quantitative reverse transcription-PCR in the wild type and cepI and cepR mutants. The expression levels of all nine genes were similar in the cepI and cepR mutants and consistent with the promoter-lux reporter activity. The expression of four selected cepIR-regulated gene promoters was examined in a cciIR mutant, and two of these promoters were also regulated by cciIR. This study extends our understanding of genes whose expression is influenced by cepIR and indicates the global regulatory effect of the cepIR system in B. cenocepacia.