Matrix metalloproteinase-3 (MMP-3) over-expression is associated with tissue destruction in the context of chronic inflammation. Previous studies showed that IL-4 inhibits induction of MMP-3 by IL-1β, and suggested that AP-1 might be involved. Here we show that IL-1 induced binding of transcription factor AP-1 to the MMP-3 promoter consists primarily of c-Jun, JunB, and c-Fos and that binding of c-Jun and c-Fos is inhibited by the combination of cytokines while binding of Jun B is not. Mutation of the AP-1 site in the MMP-3 promoter decreased the ability of IL-4 to inhibit its transcription in transfected MG-63 cells. Western blotting showed that both cytokines activate Jun N-terminal kinase (JNK), but with somewhat different kinetics, and that activation of JNK by both cytokines individually is inhibited by the combination. These results indicate that IL-4 inhibition of MMP-3 expression is associated with reduction of IL-1 induced binding of active forms of the AP-1 dimer, while less active JunB-containing dimers remain, and suggest that these changes are associated with decreased activation of JNK.

A 5T/6T polymorphism in the human MMP-3 promoter affects gene expression and impacts the risk and/or severity of various pathological conditions. Chromatin immunoprecipitation (ChIP) in human fibroblasts homozygous for the 6T site demonstrate that it is bound by NF-κB and ZBP-89 transcription factors in its native chromatin. ChIP in COS-1 cells transfected with plasmids containing the 5T and 6T sites in the context of 2 kb of the MMP-3 promoter showed that NF-κB p50 binds preferentially to the 6T site, while more ZBP-89 binding is detected to the 5T site. Over-expressed ZBP-89 increased transcription from the 5T promoter but not from the 6T, while NF-κB decreased transcription from both promoters, even in the presence of excess ZBP-89. A model is suggested in which the physiological impact of the polymorphism is dependent on the relative levels and activities of these competing factors in various cell types and conditions.

Background: Interleukin 4 (IL-4) has been shown to suppress interleukin-1 (IL-1) induced expression of matrix metalloproteinase-3 (MMP-3) in human synovial and gingival fibroblasts, but the mechanism of suppression has not been determined. Activators of peroxisome proliferatoractivated receptor-γ (PPARγ) have been shown to inhibit cytokine induced expression of MMPs in other cell types, and IL-4 has been shown to activate PPARγ by stimulating production of ligands through the lipoxygenase pathway. It has been suggested that PPARγ may inhibit expression of MMPs by competing with transcription factor AP-1 for binding to a putative composite binding element in the promoters. The objective of this study was to determine whether the suppressive effects of IL-4 on the IL-1 induced expression of MMP-3 involve activation of lipoxygenase and/or PPARγ.

Amphiphilic Janus particles self-assemble into complex metastructures, but little is known about how their assembly might be modified by weak interactions with a nearby biological membrane surface. Here, we report an integrated experimental and molecular dynamics simulation study to investigate the self-assembly of amphiphilic Janus particles on a lipid membrane. We created an experimental system in which Janus particles are allowed to self-assemble in the same medium where zwitterionic lipids form giant unilamellar vesicles (GUVs). Janus particles spontaneously concentrated on the inner leaflet of the GUVs. They exhibited biased orientation and heterogeneous rotational dynamics as revealed by single particle rotational tracking. The combined experimental and simulation results show that Janus particles concentrate on the lipid membranes due to weak particle-lipid attraction, whereas the biased orientation of particles is driven predominantly by inter-particle interactions. This study demonstrates the potential of using lipid membranes to influence the self-assembly of Janus particles.

Zinc-binding protein-89 (ZBP-89; ZNF148, BERF-1, BFCOL-1) is a zinc-finger transcription factor of the Kruppel family. It has been shown to regulate the expression of a number of genes, acting as either an activator or repressor of gene expression, depending on the context. It is over-expressed in several cancers, but has been shown to be involved in apoptosis and to have a negative influence on cell growth in part by interactions with p53. Previously, ZBP-89 was shown to activate transcription of the matrix metalloproteinase-3 (MMP-3) gene by binding to a polymorphic promoter element in competition with nuclear factor κB (NF-κB). NF-κB is known to be a key regulator of the inflammatory response, but relatively little is known about regulation of ZBP-89. In order to ascertain whether ZBP-89 is regulated during inflammation, we designed experiments to determine whether and to what extent ZBP-89 levels are affected by inflammatory cytokines. Here we show that ZBP-89 mRNA and protein expression are significantly inhibited in human fibroblasts by the inflammatory cytokine interleukin-1β. Since any change in the levels of ZBP-89 would presumably impact the regulation of MMP-3 and other ZBP-89 target genes, these results provide important insight into mechanisms involved in fine-tuning the immune response.

Periodontitis is the most common cause of adult tooth loss in the U.S., with an estimated 1 in 3 adults suffering from some form and 10 to 15% of adults developing severe forms. In addition to its direct impact, periodontitis also contributes to the development of several other diseases, including cardiovascular disease, pre‐term low birth weight, diabetes and oral cancer Since smoking and diabetes are frequently co‐existing, modifiable risk factors for the development of periodontitis, as well as for cardiovascular disease and some types of cancer, this study is designed to determine whether and to what extent these factors interact to affect the production of proteins and products that are involved in the pathogenesis of these conditions. HGF cultures derived from patients with periodontitis were incubated with IL‐1, LPS, nicotine and glucose in various combinations for 24 hours, and the levels of matrix metalloproteinase 1 and 3 in conditioned media were measured by ELISA. Results show that while nicotine alone has no effect on MMP‐3 protein expression, it acts synergistically with IL‐1 or LPS to increase its expression further. Glucose alone also has no effect on basal or IL‐1 induced MMP‐3 expression but counteracts the effects of nicotine. Similar effects were seen with MMP‐1. Further experiments are planned to determine the effects of nicotine and glucose on these and other mediators of inflammation.

Periodontitis is the most common cause of adult tooth loss in the U.S., with an estimated 1 in 3 adults suffering from some form and 10–15% of adults developing severe forms. In addition to its direct impact, periodontitis also contributes to the development of several other diseases, including cardiovascular disease, pre‐term low birth weight, and diabetes. Although the primary function of HO‐1 is the breakdown of heme to carbon monoxide, iron and bilirubin, it has also been shown to play an important role in wound repair and the resolution of inflammation by mechanisms involving homeostatic regulation of the redox state of cells. A series of experiments has been designed to determine whether and to what extent the levels of HO‐1 mRNA and protein are regulated by inflammatory cytokines in HGF isolated from individuals with or without periodontitis. Preliminary results show that HO‐1 mRNA is expressed in HGF cultures derived from patients with periodontitis and that mRNA levels are inhibited over 60% by Interleukin‐1 at 6 hours (10 ng/ml, p < 0.001). Interestingly however, HO‐1 protein levels as measured by ELISA are not decreased by IL‐1. Experiments are currently underway to address this apparent paradox, as well as the potential role of HO‐1 in regulation of inflammatory mediators in HGF.

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