Testosterone Inhibits Secretion of the Pro-Inflammatory Chemokine CXCL1 from Astrocytes.

Astrocytes play an important role in the regulation of the inflammatory response in the CNS, e.g., in demyelinating diseases. Since the chemokine CXCL1 is known to be secreted by astrocytes and to have a pro-inflammatory effect on immune cells in the CNS, we verified the effect of testosterone on its secretion in vitro (in the astrocytic cell line DI TNC1). Testosterone reduced the increase in CXCL1 production caused by the pro-inflammatory agent lysophosphatidylcholine and restored the basal production level of CXCL1. The androgen receptor (present and functional in the studied cell line) was strongly suggested to mediate this effect-its non-steroid ligand flutamide exerted an agonist-like effect, mimicking the activity of testosterone itself on CXCL1 secretion. This novel mechanism has important implications for the known immunomodulatory effect of testosterone and potentially other androgenic hormones. It provides a potential explanation on the molecular level and shows that astrocytes are important players in inflammatory homeostasis in the CNS and its hormonal regulation. Therefore, it suggests new directions for the development of the therapeutic intervention.

Whole grain metabolite 3,5-dihydroxybenzoic acid is a beneficial nutritional molecule with the feature of a double-edged sword in human health: a critical review and dietary considerations.

Nonprocessed foodstuffs of plant origin, especially whole-grain cereals, are considered to be health-promoting components of the human diet. While most of their well-studied effects derive from their high fiber content and low glycemic index, the presence of underrated phenolic phytonutrients has recently been brought to the attention of nutritionists. In this review, we report and discuss findings on the sources and bioactivities of 3,5-dihydroxybenzoic acid (3,5-DHBA), which is both a direct dietary component (found, e.g., in apples) and, more importantly, a crucial metabolite of whole-grain cereal-derived alkylresorcinols (ARs). 3,5-DHBA is a recently described exogenous agonist of the HCAR1/GPR81 receptor. We concentrate on the HCAR1-mediated effects of 3,5-DHBA in the nervous system, on the maintenance of cell stemness, regulation of carcinogenesis, and response to anticancer therapy. Unexpectedly, malignant tumors take advantage of HCAR1 expression to sense 3,5-DHBA to support their growth. Thus, there is an urgent need to fully identify the role of whole-grain-derived 3,5-DHBA during anticancer therapy and its contribution in the regulation of vital organs of the body via its specific HCAR1 receptor. We discuss here in detail the possible consequences of the modulatory capabilities of 3,5-DHBA in physiological and pathological conditions in humans.

Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines.

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles.

CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood-Brain Barrier Models.

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood-brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood-brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells.

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.

Direct Measurement of Kinetic Parameters of ABCG2-Dependent Transport of Natural Flavonoids Using a Fluorogenic Substrate.

Flavonoids are an important part of the human diet since plant-derived polyphenols and the mechanisms governing their pharmacokinetics are important both due to their own nutriceutical activity and the potential for food-drug interactions. A central determinant of absorption and distribution of flavonoids in the human body is the ATP-binding cassette transporter ABCG2, expressed in gut epithelium and other barrier tissues. While flavonoids were previously identified as substrates and/or inhibitors of this protein, precise enzyme kinetic calculations of affinity and activity parameters are rare due to the lack of suitable experimental models. We present a novel method that allows the direct measurement of kinetic constants for ABCG2-mediated cellular efflux of natural flavonoids thanks to the application of fluorogenic 2-aminoethyl diphenylborinate, which reacts with intracellular flavonoids forming a fluorescent, nonmembrane-permeable conjugate, thus making it possible to measure the intracellular substrate concentration throughout the experiment. Our studies were performed in Madin-Darby canine kidney II-derived cell lines expressing human ABCG2 and involve substrate efflux from whole, unmodified cells, precluding the need for plasma membrane vesicle preparation. We present methods for calculation of enzyme kinetic constants by measuring substrate concentration at efflux-influx equilibrium or during efflux from preloaded cells, and we obtained K m values of 137 µM for quercetin, 36 µM for kaempferol, and 348 µM for luteolin. Our method also allows direct verification of the transport inhibition mechanism and potentially the structure-activity relationship in substrates. SIGNIFICANCE STATEMENT: The study presents the first direct calculation of kinetic constants for enzyme-mediated active transport of natural flavonoids in a whole-cell assay, using a fluorogenic compound to measure intracellular substrate concentrations at specific time points. It has implications for nutriceutical use of polyphenols, mechanisms of food-drug interactions, and studies on absorption/distribution-determining membrane transporters, allowing a quantitative approach to pharmacokinetics of flavonoid transport across barrier tissues.

Fludarabine-Specific Molecular Interactions with Maltose-Modified Poly(propyleneimine) Dendrimer Enable Effective Cell Entry of the Active Drug Form: Comparison with Clofarabine.

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11.

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11ß1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

Terminal Sugar Moiety Determines Immunomodulatory Properties of Poly(propyleneimine) Glycodendrimers.

Poly(propyleneimine) dendrimers fully surface-modified with disaccharide moieties (maltose, cellobiose, and lactose) designed to mimic natural lectin receptor ligands were tested for their bioactivity in two myeloid cell lines: THP-1 and HL-60. Depending on the sugar modification, we observed variable activation of NF-κB, AP-1, and NF-AT signaling pathways: lactose-coated dendrimers had the strongest impact on marker gene expression and most signaling events with the notable exception of NF-κB activation in THP-1 cells. The two cell lines showed an overall similar pattern of transcription factor and gene expression activation upon treatment with glycodendrimers, suggesting the involvement of galectin and C-type lectin receptor types. An important result of this action was the overexpression of CD40 and IL8 genes, potentially leading to an activated, proinflammatory phenotype in the monocyte/macrophage cell lineage. These pharmacodynamic characteristics of glycodendrimers need to be taken into account during their pharmaceutical applications both in drug delivery and direct immunomodulation.

Glycodendrimer Nanocarriers for Direct Delivery of Fludarabine Triphosphate to Leukemic Cells: Improved Pharmacokinetics and Pharmacodynamics of Fludarabine.

Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter.

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.

Mechanisms of Internalization of Maltose-Modified Poly(propyleneimine) Glycodendrimers into Leukemic Cell Lines.

Poly(propyleneimine) dendrimers of fourth generation partially modified with maltose (open shell structure, PPI-m OS) have been proposed as carriers for nucleotide anticancer drugs. The aim of this work was to provide basic insight into interactions between fluorescently labeled PPI-m dendrimer and two distinct leukemia cell models: CCRF-1301 lymphoid cell line and HL-60 myeloid cell line. We applied qualitative confocal imaging and quantitative flow cytometry, as well as trypan blue quenching and pharmacological inhibition, to investigate the course, kinetics, and molecular mechanisms of internalization of nanoparticles. CCRF-1301 cells take up glycodendrimer macromolecules via a relatively slow, adsorptive endocytosis process, which is cholesterol-dependent, clathrin- and caveolin-independent, and not followed by recycling or exocytosis. Morphological features of this phenomenon point to the involvement of aggregation-induced cell polarity changes (capping). In HL-60 cells, internalization is very fast, independent of binding to the cell surface, and proceeds from the fluid phase via a classical clathrin-dependent mechanism, ending up in an endolysosomal compartment from which it is not further released. This substantial difference in internalization rate and mechanism between two cell types has important repercussions for potential application of this class of glycodendrimers as drug delivery agents.

Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line.

PURPOSE: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. METHODS: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. RESULTS: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 µM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25-100 µM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. CONCLUSION: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).

Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods.

Regulation of the human ABCB10 gene by E2F transcription factors.

Here, we report for the first time a functional study of transcriptional regulation of the human ABCB10 gene. We cloned a functional promoter sequence, and then showed that E2F2, E2F3 and E2F4 can activate transcription from the ABCB10 promoter. We identified sites responsible for this activation and confirmed direct binding of E2F4 to these sites in EMSA and ChIP assays. Finally, by silencing the expression of E2F factors we demonstrated their importance in maintenance of the basal ABCB10 expression. This study provides important atypical examples of E2F4 being a transcriptional activator rather than repressor as well as directly binding to a promoter and regulating it through an alternative and classical DNA consensus response element sequences. It also provides a mechanistic link between E2F4 and ABCB10, both of which are involved in the same physiological phenomena: erythroid lineage differentiation and maturation as well as protection against cardiomyocyte cell death.

Cellular and physiological mechanisms of halogenated and organophosphorus flame retardant toxicity.

Flame retardants (FRs) are chemical substances used to inhibit the spread of fire in numerous industrial applications, and their abundance in modern manufactured products in the indoor and outdoor environment leads to extensive direct and food chain exposure of humans. Although once considered relatively non-toxic, FRs are demonstrated by recent literature to have disruptive effects on many biological processes, including signaling pathways, genome stability, reproduction, and immune system function. This review provides a summary of research investigating the impact of major groups of FRs, including halogenated and organophosphorus FRs, on animals and humans in vitro and/or in vivo. We put in focus those studies that explained or referenced the modes of FR action at the level of cells, tissues and organs. Since FRs are highly hydrophobic chemicals, their biophysical and biochemical modes of action usually involve lipophilic interactions, e.g. with biological membranes or elements of signaling pathways. We present selected toxicological information about these molecular actions to show how they can lead to damaging membrane integrity, damaging DNA and compromising its repair, changing gene expression, and cell cycle as well as accelerating cell death. Moreover, we indicate how this translates to deleterious bioactivity of FRs at the physiological level, with disruption of hormonal action, dysregulation of metabolism, adverse effects on male and female reproduction as well as alteration of normal pattern of immunity. Concentrating on these subjects, we make clear both the advances in knowledge in recent years and the remaining gaps in our understanding, especially at the mechanistic level.

Mesalazine-PAMAM Nanoparticles for Transporter-Independent Intracellular Drug Delivery: Cellular Uptake and Anti-Inflammatory Activity.

Background: Mesalazine is one of the main drugs used to treat inflammatory bowel diseases. However, its applicability is limited by its rapid inactivation and removal from the organism, as well as the need for its membrane transporter-dependent cellular uptake to exert therapeutic effect. The present study involved the development of an innovative nanocarrier, based on poly(amidoamine) (PAMAM) dendrimer of the 4th generation, to obtain higher concentrations of the drug in the intestinal epithelial cells, thus increasing its anti-inflammatory potential. The work involved synthesis and in vitro characterization of covalent PAMAM-mesalazine conjugate with succinic linker. Results: PAMAM-mesalazine conjugate was synthesized and characterized by 1H NMR, 13C NMR, FTIR and MALDI-TOF MS. This allowed to confirm the purity of the obtained compound and intermediates. Based on the analyses, it was found that ~45 drug molecules were successfully attached to one molecule of PAMAM dendrimer. The conjugate was then characterized in terms of hydrodynamic diameter, zeta potential, spectral properties, drug release from the carrier, as well as cellular uptake and cytotoxicity in two in vitro models of gastrointestinal epithelium (CaCo-2 and HT-29 human cell lines). Analyzing cellular parameters related to the specific mechanism of action of mesalazine (inhibition of NF-κB signaling, decrease in interleukin and prostaglandin synthesis, and ROS scavenging), we showed that such a dendrimer-based carrier may enhance cellular uptake of the drug, which translated into its improved anti-inflammatory efficacy. Conclusion: The use of PAMAM macromolecule as a carrier for mesalazine increases the bioavailability of the drug, ensuring enhanced cellular uptake and bypassing the need to utilize mesalazine-specific membrane transporters. All these characteristics translate into an improved anti-inflammatory activity of mesalazine in vitro. In conjunction with appropriately designed in vivo studies, such a compound may prove to be a promising alternative to the therapeutics currently used in inflammatory bowel diseases.

Reduced Corneal Sensitivity With Neuronal Degeneration is a Novel Clinical Feature in Wolfram Syndrome.

PURPOSE: To evaluate corneal sensitivity and corneal nerve morphology among patients with Wolfram syndrome (WFS). DESIGN: An observational clinical case series with confirmatory experiments. METHODS: We included a group of 12 patients with biallelic mutations in the WFS1 gene and a control group composed of 30 individuals with type 1 diabetes (T1D). All participants (n = 42) underwent a complete ophthalmic examination, esthesiometry, and retinal nerve fiber layer assessment using optical coherence tomography. Morphologic assessment of corneal neuropathy by in vivo corneal confocal microscopy was conducted in 11 patients with WFS (both eyes) and 1 WFS patient (1 eye) as well as in 24 patients with T1D (both eyes in 6 patients and 1 eye in 18 patients). Additionally, corneas from Wfs1KO mice and their wild-type littermates were subjected to laser scanning confocal microscopy. RESULTS: Corneal sensitivity was significantly reduced in patients with WFS compared with patients with T1D (4.50 cm [interquartile range, 3.50-5.50 cm] vs 6.00 cm [interquartile range, 6.00-6.00 cm]; P < 10-5). Additionally, corneal nerve fiber and branch density as well as nerve fiber length were low among patients with WFS. Corneal sensitivity correlated with macular average thickness (R = 0.6928; P = .039) and best-corrected visual acuity (R = -0.61; P = .002) in the WFS group. Similarly, Wfs1 knockout mice also presented corneal neurodegeneration changes when corneal nerve fiber density and length were measured using laser scanning confocal microscopy. CONCLUSIONS: Decreased corneal sensitivity and corneal nerve degeneration are observed in WFS. Corneal sensitivity is linked with the degree of disease progression as measured by visual acuity and retinal thinning.

Cyanobacterial cell-wall components as emerging environmental toxicants - detection and holistic monitoring by cellular signaling biosensors.

Cyanobacterial blooms constitute a recognized danger to aquatic environment and public health not only due to presence of main group of cyanotoxins, such as microcystins, cylindrospermopsin or anatoxin-a, but also other emerging bioactivities. An innovative approach identifying such bioactivities is the application of cellular biosensors based on reporter genes which detect the impact of cyanobacterial cells and components on actual human cells in a physiological-like setting. In the present study biosensor cell lines detecting four different types of bioactivities (ARE - oxidative stress, NFKBRE - immunomodulatory pathogen-associated molecular patterns, AHRE - persistent organic pollutants, GRE - endocrine disruptors) were exposed to concentrated cyanobacterial cells from 21 environmental bloom samples and from eight cultures (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii and Raphidiopsis raciborskii). The AHRE and GRE biosensors did not detect any relevant bioactivity. In turn, ARE biosensors were significantly activated by bloom samples from Jeziorsko (180-250%) and Sulejów (250-400%) reservoirs with the highest cyanobacterial biomass, while activation by cultures was weak/undetectable. The same biosensors were stimulated by microcystin-LR (250%) and anatoxin-a (150%). The NFKBRE biosensors were activated to varying extent (140-650%) by most bloom and culture samples, pointing to potential immunomodulatory toxic effects on humans. Lipopolysaccharide and lipoproteins were identified as responsible for NFKBRE activation (probably via pattern recognition receptors), while peptidoglycan had no bioactivity in this assay. Thus, the holistic approach to sample analysis with the application of cellular biosensors geared towards 4 separate pathways/bioactivities was validated for identification of novel bioactivities in organisms with recognized public health significance (e.g. this study is the first to describe cyanobacterial lipoproteins as potential environmental immunomodulators). Moreover, the ability of cellular biosensors to be activated by intact cyanobacterial cells from blooms provides proof of concept of their direct application for environmental monitoring, especially comparison of potential threats without need for chemical analysis and identification of toxicants.

The ERK1/2-hepatocyte nuclear factor 4alpha axis regulates human ABCC6 gene expression in hepatocytes.

ABCC6 mutations are responsible for the development of pseudoxanthoma elasticum, a rare recessive disease characterized by calcification of elastic fibers. Although ABCC6 is mainly expressed in the liver the disease has dermatologic, ocular, and cardiovascular symptoms. We investigated the transcriptional regulation of the gene and observed that hepatocyte growth factor (HGF) inhibits its expression in HepG2 cells via the activation of ERK1/2. Similarly, other factors activating the cascade also inhibited ABCC6 expression. We identified the ERK1/2 response element in the proximal promoter by luciferase reporter gene assays. This site overlapped with a region conferring the tissue-specific expression pattern to the gene and with a putative hepatocyte nuclear factor 4alpha (HNF4alpha) binding site. We demonstrated that HNF4alpha regulates the expression of ABCC6, acts through the putative binding site, and determines its cell type-specific expression. We also showed that HNF4alpha is inhibited by the activation of the ERK1/2 cascade. In conclusion we describe here the first regulatory pathway of ABCC6 expression showing that the ERK1/2-HNF4alpha axis has an important role in regulation of the gene.