Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purß) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purß with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purß. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purß point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purß. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purß for ssDNA and diminished the interaction of Purß with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purß to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purß.

Purine-rich element binding protein B attenuates the coactivator function of myocardin by a novel molecular mechanism of smooth muscle gene repression.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purß) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purß to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purß specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purß homodimer. DNA binding analyses demonstrated that Purß interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purß associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purß point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purß repressor and the myocardin-serum response factor-CArG complex.

Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community.

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

Insulin-Like Growth Factor II (IGF-II) Inhibits IL-1ß-Induced Cartilage Matrix Loss and Promotes Cartilage Integrity in Experimental Osteoarthritis.

Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1ß-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1ß, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro. In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA.

Characterizing Biofilm Interactions between Ralstonia insidiosa and Chryseobacterium gleum.

Ralstonia insidiosa and Chryseobacterium gleum are bacterial species commonly found in potable water systems, and these two species contribute to the robustness of biofilm formation in a model six-species community from the International Space Station (ISS) potable water system. Here, we set about characterizing the interaction between these two ISS-derived strains and examining the extent to which this interaction extends to other strains and species in these two genera. The enhanced biofilm formation between the ISS strains of R. insidiosa and C. gleum is robust to starting inoculum and temperature and occurs in some but not all tested growth media, and evidence does not support a soluble mediator or coaggregation mechanism. These findings shed light on the ISS R. insidiosa and C. gleum interaction, though such enhancement is not common between these species based on our examination of other R. insidiosa and C. gleum strains, as well as other species of Ralstonia and Chryseobacterium. Thus, while the findings presented here increase our understanding of the ISS potable water model system, not all our findings are broadly extrapolatable to strains found outside of the ISS. IMPORTANCE Biofilms present in drinking water systems and terminal fixtures are important for human health, pipe corrosion, and water taste. Here, we examine the enhanced biofilm of cocultures for two very common bacteria from potable water systems: Ralstonia insidiosa and Chryseobacterium gleum. While strains originally isolated on the International Space Station show enhanced dual-species biofilm formation, terrestrial strains do not show the same interaction properties. This study contributes to our understanding of these two species in both dual-culture and monoculture biofilm formation.

Human Growth Factor/Immunoglobulin Complexes for Treatment of Myocardial Ischemia-Reperfusion Injury.

Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor 2 (FGF2) are receptor tyrosine kinase agonists that promote cell survival after tissue injury and angiogenesis, cell proliferation and migration during tissue repair and regeneration. Both ligands have potential as systemic treatments for ischemia-reperfusion injury, however clinical use of HGF and FGF2 has been limited by poor pharmacokinetic profiles, i.e., their susceptibility to serum proteases, rapid clearance and short half-lives. Previously, we reported vaso- and cardioprotective protein complexes formed between HGF and polyclonal, non-specific immunoglobulin (IgG) with therapeutic efficacy in a rat model of myocardial ischemia with reperfusion (MI/R). Here, using a pre-clinical porcine MI/R model, we demonstrate human HGF/IgG complexes provide significant myocardial salvage, reduce infarct size, and are detectable in myocardial tissue 24 h after intracoronary injection. Furthermore, we show that multiple daily infusions of HGF/IgG complexes after MI do not lead to production of HGF-specific auto-antibodies, an important concern for administered biologic drugs. In experiments to identify other growth factors that non-covalently interact with IgG, we found that human FGF2 associates with IgG. Similar to human HGF/IgG complexes, FGF2/IgG complexes protected primary human cardiac endothelial cells under simulated ischemia (1% oxygen and nutrient deprivation) for 48-72 h. Molecular modeling studies suggested that FGF2 and HGF both interact with the Fc domain of IgG. Also, we tested whether an Fc-fusion protein would bind FGF2 to form complexes. By native gel electrophoretic assays and biochemical pulldowns, we found that Jagged1, a Notch1 ligand that controls stem cell self-renewal and tissue regeneration, bound FGF2 when presented as a Jagged1- Fc fusion protein. Our results suggest that human growth factor/IgG and FGF2/Fc- fusion complexes have potential to provide a biologics platform to treat myocardial ischemia-reperfusion and other forms of tissue injury.

Wnt7a Inhibits IL-1ß Induced Catabolic Gene Expression and Prevents Articular Cartilage Damage in Experimental Osteoarthritis.

Wnt7a is a protein that plays a critical role in skeletal development. However, its effect on cartilage homeostasis under pathological conditions is not known. In this study, we found a unique inverse correlation between Wnt7a gene expression and that of MMP and IL-1ß in individual human OA cartilage specimens. Upon ectopic expression in primary human articular chondrocytes, Wnt7a inhibited IL-1ß-induced MMP and iNOS gene expression. Western blot analysis indicated that Wnt7a induced both canonical Wnt signaling and NFAT and Akt non-canonical signaling. Interestingly, inhibiting the canonical and Akt pathway did not affect Wnt7a activity. However, inhibiting the NFAT pathway impaired Wnt7a's ability to inhibit MMP expression, suggesting that Wnt7a requires NFAT signaling to exert this function. In vivo, intraarticular injection of lentiviral Wnt7a strongly attenuated articular cartilage damage induced by destabilization of the medial meniscus (DMM) OA-inducing surgery in mice. Consistently, Wnt7a also inhibited the progressive increase of joint MMP activity in DMM animals. These results indicate that Wnt7a signaling inhibits inflammatory stimuli-induced catabolic gene expression in human articular chondrocytes and is sufficient to attenuate MMP activities and promote joint cartilage integrity in mouse experimental OA, demonstrating a novel effect of Wnt7a on regulating OA pathogenesis.

The Chondroprotective Role of Erythromycin in a Murine Joint Destruction Model.

OBJECTIVE: Inflammation is a major player in the joint destruction process. Macrolide antibiotics have recently been found to have a novel anti-inflammatory function, but their effects on the joint are unknown. Our objective was to investigate the effect of macrolide antibiotic erythromycin on cartilage gene expression under inflammatory conditions as well as on joint pathology in an in vivo inflammatory joint destruction model. DESIGN: In our in vivo studies, mouse knee joints were injected with monosodium iodoacetate (MIA), a chemical that inhibits glycolysis and causes joint inflammation and matrix loss. Erythromycin was administered by daily intraperitoneal injection. Changes in joint cartilage and synovium were evaluated by histological analysis. In our in vitro studies, primary bovine articular chondrocytes were treated with erythromycin in the presence of pro-inflammatory cytokine IL-1ß or lipopolysaccharide (LPS), and cartilage gene expression analysis was performed. RESULTS: Regional differences in cartilage matrix destruction along the medial-lateral axis were observed in joints of MIA-injected mice. Erythromycin treatment inhibited cartilage matrix loss and synovitis in these joints. In addition, erythromycin inhibited IL-1ß and LPS-induced expression of MMPs and iNOS, as well as the positive regulatory loop between IL-1ß and Toll-like receptor 4 (TLR4) in articular chondrocytes. Furthermore, erythromycin prevented LPS-induced NF-κB activation, a key mediator of TLR4-mediated cartilage destruction process. CONCLUSIONS: Erythromycin has the ability to inhibit catabolic gene expression mediated by IL-1ß and TLR4 in chondrocytes in vitro and maintains cartilage matrix levels in experimental inflammatory joint destruction in vivo, suggesting that it possesses a chondroprotective activity.

Analysis of the trajectory of osteoarthritis development in a mouse model by serial near-infrared fluorescence imaging of matrix metalloproteinase activities.

OBJECTIVE: A major hurdle in osteoarthritis (OA) research is the lack of sensitive detection and monitoring methods. It is hypothesized that proteases, such as matrix metalloproteinases (MMPs), are up-regulated in the early stages of OA development. This study was undertaken to investigate if a near-infrared (NIR) fluorescent probe activated by MMPs could visualize in vivo OA progression beginning in the early stages of the disease. METHODS: Using an MMP-activatable NIR fluorescent probe (MMPSense 680), we assessed the up-regulation of MMP activity in vitro by incubating human chondrocytes with the proinflammatory cytokine interleukin-1ß (IL-1ß). MMP activity was then evaluated in vivo serially in a mouse model of chronic, injury-induced OA. To track MMP activity over time, mice were imaged 1-8 weeks after OA-inducing surgery. Imaging results were correlated with histologic findings. RESULTS: In vitro studies confirmed that NIR fluorescence imaging identified enhanced MMP activity in IL-1ß-treated human chondrocytes. In vivo imaging showed significantly higher fluorescence intensity in OA knees compared to sham-operated (control) knees of the same mice. Additionally, the total emitted fluorescence intensity steadily increased over the entire course of OA progression that was examined. NIR fluorescence imaging results correlated with histologic findings, which showed an increase in articular cartilage structural damage over time. CONCLUSION: Imaging of MMP activity in a mouse model of OA provides sensitive and consistent visualization of OA progression, beginning in the early stages of OA. In addition to facilitating the preclinical study of OA modulators, this approach has the potential for future translation to humans.