Eye enucleation activates the transcription nuclear factor kappa-B in the rat superior colliculus

Ocular enucleation produces significant morphological and physiological changes in central visual areas. However, our knowledge of the molecular events resulting from eye enucleation in visual brain areas remains elusive. We characterized here the transcription nuclear factor kappa-B (NF-kappa B) activation induced by ocular enucleation in the rat superior colliculus (SC). We also tested the effectiveness of the synthetic glucocorticoid dexamethasone in inhibiting its activation. Electrophoretic mobility shift assays to detect NF-kappa B indicated that this transcription factor is activated in the SC from 1 h to day 15 postlesion. The expression of p65 and p50 proteins in the nuclear extracts was also increased. Dexamethasone treatment was able to significantly inhibit NF-kappa B activation. These findings suggest that this transcriptional factor is importantly involved in the visual system short-term processes that ensue after retinal lesions in the adult brain. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Transcriptional effects of 1,25 dihydroxyvitamin D3 physiological and supra-physiological concentrations in breast cancer organotypic culture

Background Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)2D3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)2D3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)2D3 at concentrations that can be attained in vivo. Methods Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)2D3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)2D3 0.5nM, using RT-qPCR, western blot or immunocytochemistry. Results 1,25(OH)2D3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)2D3 near physiological concentration. Genes up-modulated by both 1,25(OH)2D3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)2D3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)2D3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)2D3 0.5nM was detected. Conclusions In breast cancer specimens a short period of 1,25(OH)2D3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)2D3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Die Regulation des Enzyms Paraoxonase-2 sowie dessen Einfluss auf die Hämatopoese

In der vorliegenden Arbeit wurden durch den Einsatz von drei unabhängigen Methoden Proteine und Faktoren identifiziert, die die PON2-mRNA-Expression beeinflussen. Anhand der erhaltenen Faktoren wurden verstärkt solche ausgewählt, die eine Rolle in der Tumorbiologie spielen. Unter Verwendung verschiedener Zellmodelle wurde schließlich der Effekt dieser Faktoren auf die PON2-Expression analysiert. Die Ergebnisse dieser Arbeit zeigen, dass die PON2-Expression in K562-Zellen durch den PI3K / Akt-Signalweg, der in vielen Tumoren übermäßig aktiviert vorliegt, gesteigert wird. Auch eine Beteiligung des Wnt / β-Catenin-Signalweges kann nicht ausgeschlossen werden. Pharmakologische Inhibitoren von GSK-3β, einer Kinase die in beiden Signalwegen involviert ist, führt zu einer Steigerung der PON2-Expression durch den Transkriptionsfaktor LEF-1. Des Weiteren wurde gezeigt, dass die Familie der FoxO-Transkriptionsfaktoren an der Regulation der PON2-Expression in K562-Zellen beteiligt sind, wenn gleich es für die jeweiligen FoxO-Isoformen Unterschiede gibt.rnIm Hinblick auf die Assoziation von PON2 mit Leukämien wurde anhand eines PON2-/--Mausmodells, der Einfluss von PON2 auf die Hämatopoese untersucht. Dabei wurden signifikante Unterschiede in einigen Stammzellkompartimenten festgestellt. Ferner scheint PON2 die Entwicklung von Erythrozyten und Thrombozyten zu beeinflussen. Dies äußert sich in einer offensichtlichen Splenomegalie, zumindest bei alten weiblichen PON2-/--Mäusen.rnAbschließend wurde zur Generierung eines konditionalen PON2-Überexpressionsmausmodells erfolgreich ein Gene-Targeting-Vektor entwickelt. Durch eine gewebe-, zeit- und zellspezifische Steigerung der PON2-Expression ist es möglich, den Effekt einer PON2-Überexpression im Hinblick auf verschiedene Erkrankungen zu untersuchen.rnBisher war wenig über die Regulation des humanen PON2 bekannt. Die Ergebnisse der vorliegenden Arbeit zeigen erstmals, durch welche Signalwege und Transkriptionsfaktoren PON2 in Leukämiezellen reguliert wird. Im Hinblick auf die Rolle von PON2 in der Tumorbiologie ist es erstmals möglich, die PON2-Expression gezielt durch die Inhibition bzw. Aktivierung der PON2-regulierenden Faktoren zu beeinflussen, und damit neue Wege in der Krebstherapie zu beschreiten. rn

Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene

Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

Reconstituted high-density lipoprotein modulates activation of human leukocytes

An anti-inflammatory effect of reconstituted High Density Lipoprotein (rHDL) has been demonstrated in atherosclerosis and in sepsis models. An increase of adhesion molecules as well as tissue factor expression on endothelial cells in response to inflammatory or danger signals are attenuated by the treatment with rHDL. Here we show the inhibitory effect of rHDL on the activation of human leukocytes in a whole blood assay as well as on monocyte-derived human dendritic cells (DC). Multiplex analysis of human whole blood showed that phytohaemagglutinin (PHA)-induced secretion of the cytokines IL-1β, IL-1RA, IL-2R, IL-6, IL-7, IL-12(p40), IL-15 and IFN-α was inhibited. Furthermore, an inhibitory effect on the production of the chemokines CCL-2, CCL-4, CCL-5, CXCL-9 and CXCL-10 was observed. Activation of granulocytes and CD14+ monocytes by PHA is inhibited dose-dependently by rHDL shown as decreased up-regulation of ICAM-1 surface expression. In addition, we found a strong inhibitory effect of rHDL on toll-like receptor 2 (TLR2)- and TLR4-mediated maturation of DC. Treatment of DC with rHDL prevented the up-regulation of cell surface molecules CD80, CD83 and CD86 and it inhibited the TLR-driven activation of inflammatory transcription factor NF-κB. These findings suggest that rHDL prevents activation of crucial cellular players of cellular immunity and could therefore be a useful reagent to impede inflammation as well as the link between innate and adaptive immunity.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

A urokinase receptor promoter element (-152/-135) bound with an AP-2-alpha-related protein and SP1 is required for the induction of gene expression by PMA and SRC

The urokinase-type plasminogen activator receptor (u-PAR) promotes extracellular matrix degradation, invasion and metastasis. A first objective of this dissertation was to identify cis-elements and trans-acting factors activating u-PAR gene expression through a previously footprinted (–148/–124) promoter region. Mobility shifting experiments on nuclear extracts of a high u-PAR-expressing colon cancer cell line (RKO) indicated Sp1, Sp3 and a factor similar to, but distinct from, AP-2α bound to an oligonucleotide spanning –152/–135. Mutations preventing the binding of the AP-2α-related factor reduced u-PAR promoter activity. In RKO, the expression of a dominant negative AP-2 (AP-2αB) diminished u-PAR promoter activity, protein and u-PAR mediated laminin degradation. Conversely, u-PAR promoter activity in low u-PAR-expressing GEO cells was increased by AP-2αA expression. PMA treatment, which induces u-PAR expression, caused an increased amount of the AP-2α-related factor-containing complex in GEO, and mutations preventing AP-2α-like and Sp1/Sp3 binding reduced the u-PAR promoter stimulation by PMA. In resected colon cancers, u-PAR protein amounts were related to the amount of the AP-2α-related factor-containing complex. In conclusion, constitutive and PMA- inducible u-PAR gene expression and -proteolysis are mediated partly through transactivation via a promoter sequence (–152/435) bound with an AP-2α-related factor and Sp1/Sp3. ^ A second interest of this dissertation was to determine if a constitutively active Src regulates the transcription of the u-PAR gene, since c-src expression increases invasion in colon cancer. Increased u-PAR protein and laminin degradation paralleling elevated Src activity was evident in SW480 colon cancer cells stably expressing a constitutively active Src (Y- c-src527F). Nuclear run-on experiments indicated that this was due largely to transcriptional activation. While transient transfection of SW480 cells with Y-c-src527F induced a u-PAR-CAT-reporter, mutations preventing Sp1-binding to promoter region –152/435 abolished this induction. Mobility shift assays revealed increased Sp1 binding to region –152/135 with nuclear extracts of Src-transfected SW480 cells. Finally, the amounts of endogenous u-PAR in resected colon cancers significantly correlated with Src-activity. These data suggest that u-PAR gene expression and proteolysis are regulated by Src, this requiring the promoter region (–152/–135) bound with Sp1, thus, demonstrating for the first time that transcription factor Sp1 is a downstream effector of Src. ^

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^

Transcriptional repression of serum amyloid A1 by AP -2 contributes to its liver -specific expression

Studies on the transcriptional regulation of serum amyloid A1 (SAA1) gene, a liver specific acute-phase gene, identified a regulatory element in its promoter that functioned to repress (SAA1) gene transcription in nonliver cells. This silencer element interacts with a nuclear protein that is detectable in HeLa cells, fibroblasts and placental tissues but not in liver or liver-derived cells. As the expression pattern of this repressor is consistent with its potential regulatory role in repressing SAA1 expression, and that many other liver gene promoters also contain this repressor binding site, we sought to investigate whether this repressor may have a broader functional role in repressing liver genes. ^ We have utilized protein purification, cell culture, transient and stable gene transfection, and molecular biology approaches to identify this protein and investigate its possible function in the regulation of (SAA1) and other liver genes. Analyses of amino acid sequence of the purified nuclear protein, and western blot and gel shift studies identified the repressor as transcription factor AP-2 or AP-2-like protein. Using transient transfection of DNA into cultured cells, we demonstrate that AP-2 can indeed function as a repressor to inhibit transcription of SAA1 gene promoter. This conclusion is supported by the following experimental results: (1) overexpression of AP-2 in hepatoma cells inhibits conditioned medium (CM)-induced expression of SAA1 promoter; (2) binding of AP-2 to the SAA1 promoter is required for AP-2 repression function; (3) one mechanism by which AP-2 inhibits SAA1 may be by antagonizing the activation function of the strong transactivator NFκB; (4) mutation of AP-2 binding sites results in derepression of SAM promoter in HeLa cells; and (5) inhibition of endogenous AP-2 activity by a dominant-negative mutant abolishes AP-2's inhibitory effect on SAM promoter in HeLa cells. In addition to the SAM promoter, AP-2 also can bind to the promoter regions of six other liver genes tested, suggesting that it may have a broad functional role in restricting the expression of many liver genes in nonliver cells. Consistent with this notion, ectopic expression of AP-2 also represses CM-mediated activation of human third component of complement 3 promoter. Finally, in AP-2-expressing stable hepatoma cell lines, AP-2 inhibits not only the expression of endogenous SAA, but also the expression of several other endogenous liver genes including albumin, α-fetoprotein. ^ Our findings that AP-2 has the ability to repress the expression of liver genes in nonliver cells opens a new avenue of investigation of negative regulation of gene transcription, and should improve our understanding of tissue-specific expression of liver genes. In summary, our data provide evidence suggesting a novel role of AP-2 as a repressor, inhibiting the expression of liver genes in nonliver cells. Thus, the tissue-specific expression of AP-2 may constitute an important mechanism contributing to the liver-specific expression of liver genes. ^