Tilman Sanchez-Elsner

Synergistic cooperation between hypoxia and transforming growth factor-beta pathways on human vascular endothelial growth factor gene expression

Signaling by transforming growth factor (TGF)-β family members is mediated by Smad proteins that regulate gene transcription through functional cooperativity and association with other DNA-binding proteins. The hypoxia-inducible factor (HIF)-1 is a transcriptional complex that plays a key role in oxygen-regulated gene expression. We demonstrate that hypoxia and TGF-β cooperate in the induction of the promoter activity of vascular endothelial growth factor (VEGF), which is a major stimulus in the promotion of angiogenesis. This cooperation has been mapped on the human VEGF promoter within a region at −1006 to −954 that contains functional DNA-binding sequences for HIF-1 and Smads. Optimal HIF-1α-dependent induction of the VEGF promoter was obtained in the presence of Smad3, suggesting an interaction between these proteins. Consistent with this, co-immunoprecipitation experiments revealed that HIF-1α physically associates with Smad3. These results demonstrate that both TGF-β and hypoxia signaling pathways can synergize in the regulation of VEGF gene expression at the transcriptional level.

Noncoding RNAs of Trithorax Response Elements Recruit Drosophila Ash1 to Ultrabithorax

Homeotic genes contain cis-regulatory trithorax response elements (TREs) that are targeted by epigenetic activators and transcribed in a tissue-specific manner. We show that the transcripts of three TREs located in the Drosophila homeotic gene Ultrabithorax (Ubx) mediate transcription activation by recruiting the epigenetic regulator Ash1 to the template TREs. TRE transcription coincides with Ubx transcription and recruitment of Ash1 to TREs in Drosophila. The SET domain of Ash1 binds all three TRE transcripts, with each TRE transcript hybridizing with and recruiting Ash1 only to the corresponding TRE in chromatin. Transgenic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and restores Ubx expression in Drosophila cells and tissues that lack endogenous TRE transcripts. Small interfering RNA–induced degradation of TRE transcripts attenuates Ash1 recruitment to TREs and Ubx expression, which suggests that noncoding TRE transcripts play an important role in epigenetic activation of gene expression.

Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II

Endoglin is an auxiliary component of the transforming growth factor-β (TGF-β) receptor system, able to associate with the signaling receptor types I (TβRI) and II (TβRII) in the presence of ligand and to modulate the cellular responses to TGF-β1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TβRI or TβRII. This study shows that full-length endoglin interacts with both TβRI and TβRII, independently of their kinase activation state or the presence of exogenous TGF-β1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TβRI interaction was different from that of endoglin/TβRII. TβRII interacts with the amino acid region 437–558 of the extracellular domain of endoglin, whereas TβRI interacts not only with the region 437–558 but also with the protein region located between amino acid 437 and the N terminus. Both TβRI and TβRII interact with the cytoplasmic domain of endoglin, but TβRI only interacts when the kinase domain is inactive, whereas TβRII remains associated in its active and inactive forms. Upon association, TβRI and TβRII phosphorylate the endoglin cytoplasmic domain, and then TβRI, but not TβRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TβRII, TβRI, and downstream Smad proteins as well as a modulation of TGF-β signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-β responses.

MicroRNA-155 modulates the pathogen binding ability of dendritic cells (DCs) by down-regulation of DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN).

MicroRNA-155 (miR-155) has been involved in the response to inflammation in macrophages and lymphocytes. Here we show how miR-155 participates in the maturation of human dendritic cells (DC) and modulates pathogen binding by down-regulating DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN), after directly targeting the transcription factor PU.1. During the maturation of DCs, miR-155 increases up to 130-fold, whereas PU.1 protein levels decrease accordingly. We establish that human PU.1 is a direct target for miR-155 and localize the target sequence for miR-155 in the 3′-untranslated region of PU.1. Also, overexpression of miR-155 in the THP1 monocytic cell line decreases PU.1 protein levels and DC-SIGN at both the mRNA and protein levels. We prove a link between the down-regulation of PU.1 and reduced transcriptional activity of the DC-SIGN promoter, which is likely to be the basis for its reduced mRNA expression, after miR-155 overexpression. Finally, we show that, by reducing DC-SIGN in the cellular membrane, miR-155 is involved in regulating pathogen binding as dendritic cells exhibited the lower binding capacity for fungi and HIV protein gp-120 when the levels of miR-155 were higher. Thus, our results suggest a mechanism by which miR-155 regulates proteins involved in the cellular immune response against pathogens that could have clinical implications in the way pathogens enter the human organism.

The Interleukin 13 (IL-13) Pathway in Human Macrophages Is Modulated by MicroRNA-155 via Direct Targeting of Interleukin 13 Receptor α1 (IL13Rα1)

Macrophages play a central role in the balance and efficiency of the immune response and are at the interface between innate and adaptive immunity. Their phenotype is a delicate equilibrium between the M1 (classical, pro-Th1) and M2 (alternative, pro-Th2) profiles. This balance is regulated by cytokines such as interleukin 13 (IL-13), a typical pro-M2-Th2 cytokine that has been related to allergic disease and asthma. IL-13 binds to IL-13 receptor α1 (IL13Rα1), a component of the Type II IL-4 receptor, and exerts its effects by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) through phosphorylation. MicroRNAs are short (∼22 nucleotide) inhibitory non-coding RNAs that block the translation or promote the degradation of their specific mRNA targets. By bioinformatics analysis, we found that microRNA-155 (miR-155) is predicted to target IL13Rα1. This suggested that miR-155 might be involved in the regulation of the M1/M2 balance in macrophages by modulating IL-13 effects. miR-155 has been implicated in the development of a healthy immune system and function as well as in the inflammatory pro-Th1/M1 immune profile. Here we have shown that in human macrophages, miR-155 directly targets IL13Rα1 and reduces the levels of IL13Rα1 protein, leading to diminished activation of STAT6. Finally we also demonstrate that miR-155 affects the IL-13-dependent regulation of several genes (SOCS1, DC-SIGN, CCL18, CD23, and SERPINE) involved in the establishment of an M2/pro-Th2 phenotype in macrophages. Our work shows a central role for miR-155 in determining the M2 phenotype in human macrophages.

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-β signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1–155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-β-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA12LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-β by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3′-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-β with possible implications in several human diseases where homeostasis of TGF-β might be altered.

Interactions of human endothelial cells with gold nanoparticles of different morphologies

The interactions between noncancerous, primary endothelial cells and gold nanoparticles with different morphologies but the same ligand capping are investigated. The endothelial cells are incubated with gold nanospheres, nanorods, hollow gold spheres, and core/shell silica/gold nanocrystals, which are coated with monocarboxy (1-mercaptoundec-11-yl) hexaethylene glycol (OEG). Cell viability studies show that all types of gold particles are noncytotoxic. The number of particles taken up by the cells is estimated using inductively coupled plasma (ICP), and are found to differ depending on particle morphology. The above results are discussed with respect to heating efficiency. Using experimental data reported earlier and theoretical model calculations which take into account the physical properties and distribution of particles in the cellular microenvironment, it is found that collective heating effects of several cells loaded with nanoparticles must be included to explain the observed viability of the endothelial cells.

Receptor-mediated interactions between colloidal gold nanoparticles and human umbilical vein endothelial cells.

A new strategy to manipulate cell operations is demonstrated, based on membrane-receptor-specific interactions between colloidal peptide-capped gold nanoparticles and human umbilical vein endothelial cells. It is shown that colloidal gold nanoparticles of similar charge and size but capped with different peptide sequences can deliberately trigger specific cell functions related to the important biological process of blood vessel growth known as angiogenesis. Specific binding of the peptide-capped particles to two endothelial-expressed receptors (VEGFR-1, NRP-1), which control angiogenesis, is achieved. The cellular fate of the functional nanoparticles is imaged and the influence of the different peptide-coated nanoparticles on the gene expression profile of hypoxia-related and angiogenic genes is monitored. The findings open up new avenues towards the deliberate biological control of cellular functions using strategically designed nanoparticles.

Laser-induced damage and recovery of plasmonically targeted human endothelial cells

Laser-induced techniques that employ the surface plasmon resonances of nanoparticles have recently been introduced as an effective therapeutic tool for destroying tumor cells. Here, we adopt a low-intensity laser-induced technique to manipulate the damage and repair of a vital category of noncancerous cells, human endothelial cells. Endothelial cells construct the interior of blood vessels and play a pivotal role in angiogenesis. The degree of damage and repair of the cells is shown to be influenced by laser illumination in the presence of gold nanoparticles of different morphologies, which either target the cellular membrane or are endocytosed. A pronounced influence of the plasmonic nanoparticle laser treatment on the expression of critical angiogenic genes is shown. Our results show that plasmon-mediated mild laser treatment, combined with specific targeting of cellular membranes, enables new routes for controlling cell permeability and gene regulation in endothelial cells.

Manipulation of in Vitro Angiogenesis Using Peptide-Coated Gold Nanoparticles

We demonstrate the deliberate activation or inhibition of invitro angiogenesis using functional peptide coated gold nanoparticles. The peptides, anchored to oligo-ethylene glycol capped gold nanospheres, were designed to selectively interact with cell receptors responsible for activation or inhibition of angiogenesis. The functional particles are shown to influence significantly the extent and morphology of vascular structures, without causing toxicity. Mechanistic studies show that the nanoparticles have the ability to alter the balance between naturally secreted pro- and anti-angiogenic factors, under various biological conditions. Nanoparticle-induced control over angiogenesis opens up new directions in targeted drug delivery and therapy.