Promoter-luciferase Reporter Constructs Research Articles

Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis. Although many positive regulators of VEGF have been identified, relatively little is known regarding the negative regulation of VEGF expression. We identified a zinc finger transcription factor, ZNF24, that may repress VEGF transcription. An inverse correlation between expression of VEGF and ZNF24 was observed in a series of independent studies. ZNF24 was up-regulated in angiogenic tumor nodules where VEGF expression is significantly decreased compared with preangiogenic nodules. In human breast carcinoma cells cultured under normoxic conditions, ZNF24 levels were significantly up-regulated whereas VEGF levels were low. In contrast, VEGF was significantly increased in hypoxic cells whereas ZNF24 was down-regulated. The same inverse correlation between ZNF24 and VEGF was also observed in 70% of matched cDNA pairs of normal and malignant tissues from human colon and breast biopsies. Overexpression of ZNF24 resulted in a significant down-regulation of VEGF, whereas silencing of ZNF24 with small interfering RNA led to increased VEGF expression. Cotransfection of ZNF24 and a VEGF promoter luciferase reporter construct in MDA-MB-231 cells resulted in a significant decrease in VEGF promoter activity. Taken together, these data suggest that ZNF24 is involved in negative regulation of VEGF and may represent a novel repressor of VEGF transcription.

A novel promoter regulates calcitonin receptor gene expression in human osteoclasts

The calcitonin receptor (CTR) is expressed in a wide variety of tissues and cell types. In bone, its expression is restricted to osteoclasts, the cells that mediate bone resorption. The human CTR ( hCTR) gene has a complex structural organization that exhibits similarity to the porcine ( pCTR) and mouse ( mCTR) CTR genes. In these species, alternative splicing of a single gene generates multiple CTR isoforms that are distributed in both tissue-specific and species-specific patterns. However, the structural organization of the 5′ putative regulatory region and transcriptional mechanisms responsible for tissue-specific expression of the different CTR isoforms are not fully defined. The present studies were undertaken to characterize the structural organization of the 5′-region of the hCTR and identify the regulatory regions involved in osteoclast-specific transcriptional activation. Analysis of mRNA prepared from human osteoclasts using reverse transcription-polymerase chain reaction (RT-PCR) and transient transfection of hCTR promoter-luciferase reporter constructs identified two regions in the 5′-flanking sequence of the hCTR gene that regulated CTR gene expression in osteoclasts. Both of these putative promoters were responsive to the osteoclast-inducing cytokine, receptor activator of NF-κB ligand (RANKL) and demonstrated trans-activation by the RANKL-induced transcription factor nuclear factor of activated T cells (NFATc1), consistent with a role in regulating CTR gene expression in osteoclasts.

Oxidative Stress Modulates Complement Factor H Expression in Retinal Pigmented Epithelial Cells by Acetylation of FOXO3

Age-related macular degeneration (AMD), the leading cause of severe vision loss in the elderly, is a complex disease that results from genetic modifications that increase susceptibility to environmental exposures. Smoking, a major source of oxidative stress, increases the incidence and severity of AMD, and antioxidants slow progression, suggesting that oxidative stress plays a major role. Polymorphisms in the complement factor H (CFH) gene that reduce activity of CFH increase the risk of AMD. In this study we demonstrate an interaction between these two risk factors, because oxidative stress reduces the ability of an inflammatory cytokine, interferon-gamma, to increase CFH expression in retinal pigmented epithelial cells. The interferon-gamma-induced increase in CFH is mediated by transcriptional activation by STAT1, and its suppression by oxidative stress is mediated by acetylation of FOXO3, which enhances FOXO3 binding to the CFH promoter, reduces its binding to STAT1, inhibits STAT1 interaction with the CFH promoter, and reduces expression of CFH. Expression of SIRT1, a mammalian homolog of NAD-dependent protein deacetylase sir2, attenuated FOXO3 recruitment to the CFH regulatory region and reversed the H(2)O(2)-induced repression of CFH gene expression. These data suggest an important interaction between environmental exposure and genetic susceptibility in the pathogenesis of AMD and, by elucidating molecular signaling involved in the interaction, provide potential targets for therapeutic intervention.

Developmental Expression and Gene Regulation of Insulin-like 3 Receptor RXFP2 in Mouse Male Reproductive Organs1

The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.

Synaptosomal-Associated Protein 25 Gene Expression Is Hormonally Regulated during Ovulation and Is Involved in Cytokine/Chemokine Exocytosis from Granulosa Cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.

The effect of P53 gene status on the interaction of vorinostat (Suberoylanilide Hydroxamic Acid-SAHA) with carboplatin in non-small cell lung cancer (NSCLC) cell lines

10567 Background: Vorinostat, a Histone Deacetylase (HDAC) inhibitor is a novel targeted antineoplastic agent with promising activity when combined with carboplatin-paclitaxel against NSCLC. The exact molecular mechanism underlying its growth inhibitory and apoptotic effects is not well understood. We investigated the influence of p53 gene status on the interaction of vorinostat and carboplatin (a DNA targeting agent) in various NSCLC cell lines. Methods: NSCLC cells with wild type p53 (A549, 128.88T), mutant p53 (201T) and p53 null phenotype (Calu-1) were used. Cytotoxicity induced by serial dilution of carboplatin in the presence and absence of a fixed dose (1μM) of vorinostat was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. In a separate experiment, cells were also transiently transfected with a p21 promoter-luciferase reporter construct to assess p53 activation following a 24 h exposure to vorinostat, carboplatin, or vorinostat/Carboplatin combination. Luciferase activity was quantified by luminometry and corrected for total protein. Results: Vorinostat displayed single agent activity in each cell line, with greater growth inhibition observed in the p53 mutant and null cells. Synergistic interactions between carboplatin and vorinostat were observed in p53 wild type cells and the IC50 for carboplatin was reduced 3- to 5-fold. In contrast, the interaction between vorinostat and carboplatin was additive or less than additive in p53 mutant and p53 null cells. Vorinostat also increased expression of the p21 reporter construct in each of the cell lines. Conclusions: Vorinostat regulates p21 gene expression and elicits anti-tumor activity in NSCLC cells independent of their p53 status. Vorinostat potentiates carboplatin-induced cytotoxicity in NSCLC with wild type p53 but not p53 deficient cells, suggesting involvement of a p53 dependent pathway. The addition of vorinostat may allow for a reduction in standard dose of carboplatin with improvement in overall therapeutic index. A phase II/III clinical trial is in progress to evaluate vorinostat in combination with carboplatin-based regimen in advanced NSCLC. Support: CA099168–01, ASCO Foundation CDA No significant financial relationships to disclose.

A Novel Pathway to Enhance Adipocyte Differentiation of 3T3-L1 Cells by Up-regulation of Lipocalin-type Prostaglandin D Synthase Mediated by Liver X Receptor-activated Sterol Regulatory Element-binding Protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Platelet-Activating Factor Overturns the Transcriptional Repressor Disposition of Sp1 in the Expression ofMMP-9in Human Corneal Epithelial Cells

Matrix metalloproteinase (MMP)-9 is induced in corneal epithelial cells stimulated with platelet-activating factor (PAF), and interferes with the normal reepithelialization of wounded cornea. Here the transcriptional regulation of MMP-9 gene expression by PAF was investigated in human corneal epithelial cells (HCECs). DNA-binding activity of NFkappaB, Sp1, and AP-1 was determined in quiescent and PAF-stimulated HCECs by electrophoretic mobility shift assay (EMSA). A series of 5' deleted human MMP-9 promoter-luciferase reporter constructs was transiently transfected into HCECs, and luciferase activity was examined after stimulation with PAF. Mutagenesis and specific deletions of some elements in the MMP-9 promoter were also introduced and analyzed. Phosphorylation of Sp1 and MEK/ERK pathway proteins was examined by Western blot analysis. Activation of Sp1 and MMP-9 was also determined by ELISA and zymography, respectively, in the absence or presence of the MEK inhibitor PD98059. DNA-binding activity of NFkappaB, Sp1, and AP-1 was upregulated by PAF with a peak at 1 hour after stimulation. A region spanning -670 to -460 relative to the transcription start point was required for the induction of the MMP-9 promoter by PAF. Mutation of the -79AP-1 or -600NFkappaB motif reduced the activity of MMP-9 promoter and the induction of gene expression by PAF. In untreated HCECs, mutation of the -558Sp1 motif upregulated gene expression, but it caused a significant decrease in the promoter activity induced by PAF. Inhibition of MEK activity eliminated the PAF-induced phosphorylation and activation of Sp1 and abolished the upregulation of MMP-9 expression and activity. These findings demonstrate that collaboration between several regulatory elements is required for the induction of MMP-9 promoter activity by PAF and that PAF overturns the repressor effect of Sp1 through activation of the MEK/ERK signaling cascade.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Helicobacterand gastrin stimulate Reg1 expression in gastric epithelial cells through distinct promoter elements

The gastric pathogen Helicobacter pylori accelerates the progression to gastric cancer but the precise mechanisms that mediate carcinogenesis remain unidentified. We now describe how Helicobacter and gastrin stimulate the expression of a putative growth factor, Reg1, in primary gastric epithelial cells. RT-PCR and Western immunoblotting of human gastric corpus and antrum showed significantly increased Reg1alpha in H. pylori-infected patients. Similarly, Reg1 was increased in the stomachs of H. felis-infected INS-GAS mice. To study transcriptional regulation of the Reg1 gene, we transfected primary mouse gastric glands with -2111 bp and -104 bp Reg1 promoter-luciferase reporter constructs. Expression of both constructs was detected in pepsinogen- and VMAT-2-expressing cells, which corresponds to the normal pattern of expression of human and mouse endogenous Reg1. The expression of both constructs was increased in response to gastrin and H. pylori, and there were potentiating interactions between them; in contrast, only the -2111 bp construct responded to H. felis. Mutation of a C-rich putative regulatory element within the -104 bp sequence abolished the response to gastrin but not to H. pylori whereas mutation of the proximal -98 to -93 region of the promoter reduced the response to H. pylori but not to gastrin. Stimulation of Reg1 by H. pylori required the virulence factor CagA. These data indicate that expression of the putative growth factor Reg1 is controlled through separate promoter elements by gastrin and Helicobacter.

Angiotensin-TGF-β1 Crosstalk in Human Idiopathic Pulmonary Fibrosis:Autocrine Mechanisms in Myofibroblasts and Macrophages

Angiotensin II (ANGII) has been identified as a proapoptotic and profibrotic factor in experimental lung fibrosis models, and patients with the ID/DD polymorphism of ANG converting enzyme (ACE), which confers higher levels of ACE, are predisposed to lung fibrosis (Hum. Pathol. 32:521-528, 2001). Previous work from this laboratory has shown that human lung myofibroblasts isolated from patients with Idiopathic Pulmonary Fibrosis (IPF) synthesize the ANGII precursor angiotensinogen (AGT) constitutively. In attempts to understand the mechanisms and consequences of constitutive AGT synthesis by myofibroblasts, we studied myofibroblast-rich primary cultures of lung fibroblasts from patients with IPF (HIPF isolates), primary fibroblasts from normal human lung (NLFs), the IMR90 and WI38 human lung fibroblasts cell lines, and paraffin sections of lung biopsies from patients with IPF. Compared to the normal NLF isolates, HIPF primary fibroblast isolates constitutively synthesized more AGT and TGF-beta1 mRNA, and released more AGT protein, ANGII and active TGF-beta1 protein into serum-free conditioned media (both p<0.01). Incubation of HIPF fibrotic isolates with the ANGII receptor antagonist saralasin reduced both TGF-beta1 mRNA and active protein, suggesting that the constitutive expression of AGT drives the higher expression of TGF-beta1 by the HIPF cells. Consistent with this premise, treatment of either the primary NLFs or the WI38 cell line with 10(-7) M ANGII increased both TGF-beta1 mRNA and soluble active TGF-beta1 protein. Moreover, induction of the myofibroblast transition in the IMR90 cell line with 2 ng/ml TGF-beta1 increased steady state AGT mRNA levels by realtime PCR (8-fold, p<0.01) and induced expression of an AGT promoter-luciferase reporter construct by over 10-fold (p<0.001). Antisense oligonucleotides against TGF-beta1 mRNA or TGF-beta neutralizing antibodies, when applied to the fibrotic HIPF cells in serum-free medium, significantly reduced AGT expression. In lung sections from IPF patient biopsies, immunoreactive AGT/ANGI proteins were detected in myofibroblasts, epithelial cells and presumptive alveolar macrophages. Together, these data support the existence of an angiotensin/TGF-beta1 "autocrine loop" in human lung myofibroblasts and also suggest ANG peptide expression by epithelia and macrophages in the IPF lung. These findings may explain the ability of ACE inhibitors and ANG receptor antagonists to block experimental lung fibrosis in animals, and support the need for evaluation of these agents for potential treatment of human IPF. This manuscript discusses the data described above and their implications regarding IPF pathogenesis.

Insulin-like Growth Factor-I Controls BRCA1 Gene Expression through Activation of Transcription Factor Sp1

The insulin-like growth factors (IGFs) have a central role in mammary gland growth and differentiation as well as in breast cancer development. The BRCA1 gene encodes a pleiotropic protein that functions as a transcription factor. Germline BRCA1 mutations are associated with inherited predisposition to breast and ovarian cancer and confer a substantially increased risk for developing these neoplasms. Several lines of evidence led us to hypothesize that there is a functional interaction between the BRCA1 and IGF-I systems relevant to breast cancer biology. The present study tested the notion that BRCA1 gene expression is regulated by the IGF-I signaling pathway. Results of Western immunoblotting and RT-PCR analyses show that IGF-I stimulates BRCA1 protein and mRNA levels. Transient transfection experiments using BRCA1 promoter-luciferase reporter constructs reveal that IGF-I enhances BRCA1 promoter activity, suggesting that the effect of IGF-I is mediated at the transcriptional level. In addition, we provide evidence that the Sp1 zinc-finger protein is directly involved in BRCA1 gene transactivation. Combined, our data suggests that, at least part of the biological actions of IGF-I in mammary gland cells may be mediated through BRCA1. Dysregulated BRCA1 expression resulting from aberrant IGF signaling may have important consequences relevant to breast cancer pathogenesis.

Direct Interaction between USF and SREBP-1c Mediates Synergistic Activation of the Fatty-acid Synthase Promoter

To understand the molecular mechanisms underlying transcriptional activation of fatty-acid synthase (FAS), we examined the relationship between upstream stimulatory factor (USF) and SREBP-1c, two transcription factors that we have shown previously to be critical for FAS induction by feeding/insulin. Here, by using a combination of tandem affinity purification and coimmunoprecipitation, we demonstrate, for the first time, that USF and SREBP-1 interact in vitro and in vivo. Glutathione S-transferase pulldown experiments with various USF and sterol regulatory element-binding protein (SREBP) deletion constructs indicate that the basic helix-loop-helix domain of USF interacts directly with the basic helix-loop-helix and an N-terminal region of SREBP-1c. Furthermore, cotransfection of USF and SREBP-1c with an FAS promoter-luciferase reporter construct in Drosophila SL2 cells results in highly synergistic activation of the FAS promoter. We also show similar cooperative activation of the mitochondrial glycerol-3-phosphate acyltransferase promoter by USF and SREBP-1c. Chromatin immunoprecipitation analysis of mouse liver demonstrates that USF binds constitutively to the mitochondrial glycerol 3-phosphate acyltransferase promoter during fasting/refeeding in vivo, whereas binding of SREBP-1 is observed only during refeeding, in a manner identical to that of the FAS promoter. In addition, we show that the synergy we have observed depends on the activation domains of both proteins and that mutated USF or SREBP lacking the N-terminal activation domain could inhibit the transactivation of the other. Closely positioned E-boxes and sterol regulatory elements found in the promoters of several lipogenic genes suggest a common mechanism of induction by feeding/insulin.

LXR Alpha Transactivates Mouse Organic Solute Transporter Alpha and Beta via IR-1 Elements Shared with FXR

Recently identified organic solute transporter (Ost) alpha and beta are located on the basolateral membrane of enterocytes and may be responsible for the intestinal absorption of many substrates including bile acids. In the present study, the mechanism governing the transcriptional regulation of their expression was investigated. To clarify the transcriptional regulation of Osts, reporter gene assays were performed using mouse Ostalpha/beta promoter-luciferase reporter constructs. Co-transfection of the constructs with farnesoid X receptor (FXR) and retinoid X receptor alpha (RXRalpha) or liver X receptor alpha (LXRalpha) and RXRalpha into Caco-2 cells induced the transcriptional activities of both Ost alpha and beta and further increases were observed following treatment with each agonist. Sequence analyses indicated the presence of IR-1 regions in Ostalpha and Ostbeta promoters, which was confirmed by the finding that the deletion of IR-1 sequences abolished the response to FXR and LXRalpha. Furthermore, mutations in IR-1 reduced the FXR- and LXRalpha-dependent transactivation of Ostalpha/beta. Together with the detection of direct binding of FXR/RXRalpha and LXRalpha/RXRalpha to the IR-1 elements, the presence of functional FXRE/LXRE was revealed in the promoter region of both Ostalpha and Ostbeta. In addition, the stimulatory effect of FXR/RXRalpha and LXRalpha/RXRalpha on Ostalpha, but not on Ostbeta, was further enhanced by HNF-4alpha. It was concluded that LXRalpha/RXRalpha transcriptionally regulate mouse Ostalpha/beta via IR-1 elements shared with FXR/RXRalpha. Exposure to FXR/LXRalpha modulators may affect the disposition of Ostalpha/beta substrates.

Glucocorticoids exacerbate hypoxia-induced expression of the pro-apoptotic gene Bnip3 in the developing cortex

Neonatal administration of the synthetic glucocorticoid, dexamethasone (DEX) retards brain growth, alters adult behaviors and induces cell death in the rat brain, thereby implicating glucocorticoids as developmentally neuroendangering compounds. Glucocorticoids also increase expression of pro-apoptotic Bcl-2 family members and exacerbate expression of hypoxic responsive genes. Bnip3 is a pro-apoptotic Bcl-2 family member that is upregulated in response to hypoxia. In these studies, we investigated the interactions of glucocorticoid receptor and hypoxia in the regulation of Bnip3 mRNA in cortical neurons. Using quantitative real time reverse transcription–polymerase chain reaction, we found that DEX treatment of postnatal days 4–6 rat pups caused a significant increase in Bnip3 mRNA expression compared with vehicle controls. A significant increase in Bnip3 mRNA was also measured in primary cortical neurons 72 h after treatment with RU28362, a glucocorticoid receptor selective agonist. In primary cortical neurons, hypoxia increased Bnip3 mRNA expression and this was exacerbated with RU28362 treatment. To elucidate the mechanism of glucocorticoid- and hypoxia-mediated regulation of Bnip3 transcription, a Bnip3 promoter–luciferase reporter construct was utilized in primary cortical neurons. Upregulation of the Bnip3 promoter was mediated by a single glucocorticoid response element and a hypoxic response element. Bnip3 overexpression in primary cortical neurons significantly increased cell death, which is dependent on the Bnip3 transmembrane domain. However, despite the increased expression of Bnip3 following glucocorticoid and hypoxia treatment, corresponding decreases in cell survival were minimal. These studies identify a novel pathway in the developing cortex through which glucocorticoids may enhance a metabolic insult, such as hypoxia.

Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2AReceptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2A receptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1alpha expression. HIF-1alpha mRNA levels were increased in LPS-treated macrophages in an NF-kappaB-dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1alpha promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1alpha promoter activity, indicating that the A2AR-dependent increase in HIF-1alpha mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1alpha protein and DNA binding levels. Deletion of putative NF-kappaB-binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-kappaB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1alpha through the HRE. HIF-1alpha is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

Molecular Mechanism of Adaphostin-mediated G1 Arrest in Prostate Cancer (PC-3) Cells

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.

Phosphatidylinositol 3-Kinase-dependent Suppression of the Human Inducible Nitric-oxide Synthase Promoter Is Mediated by FKHRL1

The synthesis of nitric oxide by inducible nitric-oxide synthase (iNOS) plays an important role in the innate immune response by promoting microbial killing and cell damage. In response to inflammatory cytokines and bacterial products, the human iNOS (hiNOS) gene undergoes rapid transcriptional activation via binding of stimulatory transcription factors (e.g. AP-1 and NF-kappaB) to its 5'-flanking region. However, maximal hiNOS promoter induction was suppressed via an unknown phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. We hypothesized that inhibition of the transcription factor FKHRL1 by the PI3K/protein kinase B pathway attenuates hiNOS promoter induction by bacterial lipopolysaccharide and interferon-gamma (LPS/IFN-gamma). Human lung epithelial adenocarcinoma (A549) cells were transiently transfected with an 8.3-kb hiNOS promoter luciferase reporter construct. Co-expression of dominant-negative protein kinase B potentiated LPS/IFN-gamma-stimulated hiNOS promoter activity. In response to LPS/IFN-gamma, FKHRL1 was phosphorylated in a PI3K- and time-dependent fashion. Co-expression of constitutively active FKHRL1 increased hiNOS promoter activity and mRNA levels. Dominant-negative siRNA expression showed that FKHRL1 was necessary for the inhibitory effects of PI3K on hiNOS induction. The same effect was observed upon mutation of a consensus FKHRL1-binding site in the hiNOS promoter. By gel-shift analysis, the corresponding oligonucleotide probe bound endogenous FKHRL1 in an LPS/IFN-gamma- and PI3K-sensitive fashion. Regulation of the hiNOS promoter by FKHRL1 represents a potentially important molecular mechanism by which the PI3K pathway might suppress pro-inflammatory and proapoptotic responses to cytokines and bacterial products.