Promoter-luciferase Reporter Gene Construct Research Articles

Mo1772 Lipopolysaccharides Can Accelerate the Development of Nonalcoholic Fatty Liver Disease, Caused by the Up-Regulation of Lipogenic Gene Expressions

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1774 Sitagliptin Can Inhibit the Acceleration of the Development in NAFLD Caused by Lipopolysaccharides Through Inhibiting Expression of MYD88 and NfκB

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Mo1771 Adiponectin Antagonises Leptin-Induced Hepcidin Expression in Human Liver Cells: New Insights Into Obesity-Associated Iron Deficiency

Background/Aim: Obesity is rapidly becoming a pandemic and is associated with increased prevalence of iron deficiency anaemia (IDA). Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related IDA. Since leptin shares a number of common biological features with IL-6, a major factor in the development of anaemia of chronic inflammation (ACI), we speculated that leptin and its metabolic counterpart adiponectin might play a role in regulating iron metabolism in the overweight population. Methods: The human hepatoma cells HuH7 and Hep G2 were exposed to IL-6 [10-50ng/ml], leptin [0.1-10μg/ml] and adiponectin [5μg/ml] for 16 hours. mRNA expression was determined using real-time quantitative RT-PCR, protein levels were detected by western blot analysis. Both cell lines were also transfected with a hepcidin promoter-luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. Results: Similar to IL-6 (***p<0.001), leptin causes a significant dose-dependent increase of hepcidin promoter activity (***p<0.001) in both cell lines, leading to elevated mRNA-levels. The activation of transcription factor STAT3 seems to play a crucial role. This was confirmed with specific STAT3-Inhibitor S3I201 [50μM] (***p<0.001). Furthermore, we were able to show for the first time that adiponectin completely abolished leptin effects on hepcidin promoter activation (***p<0.001 in HepG2, *p<0.05 in Huh7). Conclusion: Collectively, these data provide a novel insight into the molecular link between obesity and IDA and further demonstrate that adiponectin has the molecular potential to inhibit leptininduced hepcidin expression.

Abstract 1312: The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways.

Abstract Estrogen and estrogen receptors (ERα and ERβ) play an important role in breast cancer. Currently, antiestrogen (AE) targeted to ERα and aromatase inhibitors (AI), which block local and systemic estrogen production, are used in the treatment of breast cancer. Both treatments improve outcomes for about 50% of patients with early or advanced ERα positive breast cancer. Unfortunately, in women with advanced breast cancer, essentially all breast cancers develop resistance to these two classes of agents. Moreover, the side effects associated with currently used AI limit the long-term utility of them as chemopreventative agents. Therefore there is a critical need to develop effective alternate strategies to prevent or delay the development of resistance to endocrine therapy and the resulting tumor progression. Recent studies have identified several natural compounds that have the potential to function as ERβ agonists. Plant-derived ERβ-agonists, Liquiritigenin (LIQ), from the plant Glycyrrhizae uralensis and an active component of MF101, and S-equol, from the soy isoflavone daidzein, are currently being tested to treat vasomotor hot flashes associated with menopause. In addition, S-equol is also being tested as a treatment for BPH in men. However, the exact mechanism of action of these compounds is not clear. In this study we sought to determine the mechanism by which ERβ acts as a tumor suppressor, and whether these ERβ agonists could resensitize Letrozole-resistant breast cancer cells. MCF7aro and LTLT-ca cells were used as models of hormone-responsive and hormone-resistant breast cancer respectively. MTT assay was used to determine the half maximal inhibitory concentrations of the ERβ agonists. Upon treatment, our results show changes in the levels of p53, NFkB, cyclin D1, KLF5 and others using qRT-PCR and western blot analysis. The effects of ERβ agonists on the activation of these genes were confirmed using specific gene promoter-luciferase reporter gene constructs. Specificity of ERβ-mediated actions was confirmed using a siRNA approach in addition to other methods and knockdown efficiency was confirmed through RT-PCR. Interestingly, our results show that the plant-derived ERβ-agonists, LIQ and S-equol, not only affect the growth of breast cancer cells and tumors but treatment increases the expression of p53 and others, and upon ERβ knockdown this effect is ablated. This study suggests that ERβ acts as a tumor suppressor by regulating the expression of p53 and others involved in cell proliferation. In conclusion, this study demonstrates the potential role for ERβ agonists to improve adjuvant endocrine therapy to treat both hormone-responsive and hormone-resistant breast cancers, as well as provides rationale for new preventive approaches to decrease the incidence of breast cancer. Citation Format: Cathy Samayoa, Anil Kotha, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar Rao Tekmal. The estrogen receptor β agonists, Liquiritigenin and S-equol, inhibit breast cancer cell proliferation through the activation of tumor-suppressor and other pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2013-1312

Transcriptional effects of a lupus-associated polymorphism in the 5′ untranslated region (UTR) of human complement receptor 2 (CR2/CD21)

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic component that determines risk. A common three single-nucleotide polymorphism (SNP) haplotype of the complement receptor 2 (CR2) gene has been associated with increased risk of SLE (Wu et al., 2007; Douglas et al., 2009), and a less common haplotype consisting of the major allele at SNP1 and minor alleles at SNP2 and 3 confers protection (Douglas et al., 2009). SNP1 (rs3813946), which is located in the 5′ untranslated region (UTR) of the CR2 gene, altered transcriptional activity of a CR2 promoter–luciferase reporter gene construct transiently transfected into a B cell line (Wu et al., 2007) and had an independent effect in the protective haplotype (Douglas et al., 2009). In this study, we show that this SNP alters transcriptional activity in a transiently transfected non B-cell line as well as in stably transfected cell lines, supporting its relevance in vivo. Furthermore, the allele at this SNP affects chromatin accessibility of the surrounding sequence and transcription factor binding. These data confirm the effects of rs3813946 on CR2 transcription, identifying the 5′ UTR to be a novel regulatory element for the CR2 gene in which variation may alter gene function and modify the development of lupus.

Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

Transcriptional Up-Regulation by Aldosterone of the Cardiac Cav1.2 Encoding Gene CACNA1C

Although the effect of aldosterone beyond the epithelia is now well recognized, specific action through the mineralocorticoid receptor (MR) in heart is still matter of debate. In the past decade, we have accumulated evidence, both ex vivo and in vivo, that modulation of Ca2+ signaling, especially Ca2+ influx via L-type Ca2+ channel (Cav1.2), is a central factor in the cardiac action of aldosterone. Even if our results established that a specific transcriptional upregulation is involved in the effect of aldosterone on Cav1.2, it cannot be concluded whether this genomic effect is direct or indirect. The effect of the mineralocorticoid, aldosterone, on the regulation of the cardiac Cav1.2 expression was investigated in primary cultures of neonatal rat ventricular myocytes (nRVM). By use of quantitative real time RT-PCR, we found that aldosterone increased the Cav1.2 mRNA amount in time- and dose-dependent manner. This up-regulation appeared as early as 1hour of incubation even with 1 nM aldosterone. Inspection of the upstream promoter region from rat Cav1.2 encoding gene, revealed one putative hormone response element, GRE. The activity of the promoter-luciferase reporter gene constructs in transfected nRVMs showed similar time- and dose-dependent stimulation with aldosterone. We will examine the intracellular trafficking of MR in nRVMs using a fusion protein of green fluorescent protein and human MR. Selectivity of these effects will be tested as well as electrophoretic mobility shift assays to identify the region of the cacna1c promoter that can bind MRs. The data will provide new insights in the molecular mechanisms underlying the regulation of Cav1.2 channel expression by mineralocorticoids.

Transcriptional regulation of UCP4 by NF-κB and its role in mediating protection against MPP+ toxicity

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal cell survival in MPP+-induced toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential. UCP4 expression is increased by MPP+, but its regulation is unknown. Using serial human UCP4 promoter–luciferase reporter gene constructs, we identified and characterized several cis-acting elements that can regulate UCP4 expression. Core promoter activity exists within 100bp upstream of the transcription initiation site (TIS=+1). Both CAAT box (−33/−27) and Sp1 (−62/−49) elements are crucial and act synergistically in its transcription. We identified a NF-κB putative binding site at −507/−495. Mutation of this site significantly decreased UCP4 promoter activity. Activation of NF-κB by TNFα or cycloheximide increased, whereas its inhibition by 4-hydroxy-2-nonenal or transfection of pIκBαM suppressed, UCP4 promoter activity. NF-κB inhibition significantly suppressed the MPP+-induced increase in UCP4 expression. MPP+ increased specific binding of NF-κB protein complexes to this site in electrophoretic mobility shift assay. Both UCP4 knockdown and NF-κB inhibition exacerbated MPP+-induced cell death. We present the first direct evidence that UCP4 is regulated by NF-κB, mediated via a functional NF-κB site in its promoter region, and that UCP4 has a significant role in NF-κB prosurvival signaling, mediating its protection against MPP+ toxicity.

Different roles of H-ras for regulation of myosin heavy chain promoters in satellite cell-derived muscle cell culture during proliferation and differentiation

The effect of constitutively activated proto-oncogene H-ras (H-rasQ61L) on the regulation of myosin heavy chain (MHC) promoter activities was investigated in rabbit satellite cell-derived muscle cell culture during the proliferation stage and early and later stages of differentiation, respectively. During proliferation, overexpression of H-rasQ61L did not affect basal level of activity of the slow MHCI/beta or the fast MHCIId/x promoter luciferase reporter gene construct in transient transfection assays. By contrast, H-rasQ61L affected both MHC promoter activities during differentiation, and this effect changes from inactivation after 2 days to activation after 4 days of differentiation. The activating effect of H-rasQ61L on both MHC promoters after 4 days of differentiation was significantly reduced by LY-294002, a specific inhibitor of the phosphoinositol-3-kinase (PI3K), a downstream target of Ras. Furthermore, the protein kinase Akt (protein kinase B), a downstream target of PI3k, was activated 4 days after initiation of differentiation in myotubes overexpressing H-rasQ61L. By contrast, inhibition of another Ras downstream pathway, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 (MKK1/2-ERK1/2-MAPK), increased activities of both MHC promoters, indicating a suppressive role of this pathway. Moreover, the Ras-PI3K-Akt signaling pathway is involved in the activation of MHCI/beta and IId/x promoters in a later stage of differentiation of muscle cells, presumably by a known inhibiting effect of activated Akt on the MKK1/2-ERK1/2-MAPK pathway. The experiments demonstrate that during differentiation of muscle cells activated H-ras is an important regulator of MHC isoform promoter function with opposite effects during early and later stages.

Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG

Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-gamma. However, in this study we found that IFN-gamma down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-gamma resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-gamma activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-gamma, whereas expression of JAK1 or STAT-1 protein restored the IFN-gamma inhibitory effect on luciferase activity. The repressive effect of IFN-gamma on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-gamma activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-gamma stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-gamma.

Inhibitors of Inducible NO Synthase Expression: Total Synthesis of (S)‐Curvularin and Its Ring Homologues

(S)-Curvularin and its 13-, 14-, and 16-membered lactone homologues were synthesized through a uniform strategy in which a Kochi oxidative decarboxylation and ring-closing metathesis reactions constitute the key processes. In the evaluation of the anti-inflammatory effects of the synthesized compounds in assays using cells stably transfected with a human iNOS promoter-luciferase reporter gene construct, the 14- and 16-membered homologues showed a slightly higher inhibitory effect towards iNOS promoter activity than curvularin itself. However, the larger ring homologues also exhibited higher cytotoxicity, manifest in downregulated eNOS promoter activity. In contrast, the di-O-acetyl and 4-chloro derivatives of (S)-curvularin showed higher inhibitory efficiency towards induction of the iNOS promoter and less negative effect on eNOS promoter activity than curvularin.

Leptin Increases the Expression of the Iron Regulatory Hormone Hepcidin in HuH7 Human Hepatoma Cells

Obesity is a major global health problem and is associated with low-grade inflammation and, in a number of cases, poor iron status. We speculated that the adipokine leptin might play a role in regulating iron metabolism in the overweight population because it shares a number of common biological features with IL-6, a major factor in the development of the anemia of chronic disease via its stimulatory actions on the production and release of the iron regulatory hormone hepcidin. To test this hypothesis, we exposed HuH7 human hepatoma cells to leptin and measured hepcidin mRNA expression by quantitative PCR. HuH7 cells were also transfected with a hepcidin promoter–luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. In leptin-treated cells, hepcidin mRNA expression was enhanced significantly. Preincubation with a Janus kinase (JAK) 2 inhibitor significantly diminished this response. Hepcidin promoter activity was also increased in the presence of leptin. This effect was decreased either by mutation of the signal transducer and activator of transcription (STAT) 3 binding motif in the hepcidin promoter or by coexpressing a dominant-negative STAT3 mutant. These data suggest that leptin upregulates hepatic hepcidin expression through the JAK2/STAT3 signaling pathway. As a consequence, the increased production of leptin in overweight individuals might be a major contributor to the aberrant iron status observed in these population groups.

Transcriptional regulation of intronic calcium-activated potassium channel SK2 promoters by nuclear factor-kappa B and glucocorticoids

Small-conductance Ca(2+)-activated K(+) channels (SK) of the SK2 subtype are widely expressed in the central nervous system where they contribute to the control of neuronal excitability. Two SK2 isoforms, SK2-S and SK2-L, the latter representing an N-terminally extended protein of SK2-S, are expressed in similar patterns in the brain. However, our understanding of mechanisms by which the expression of SK2 is regulated is limited. We identified one functional glucocorticoid response element (GRE) at position -2248 bp and two functional nuclear factor-kappB (NF-kappaB) response elements at positions -1652 and -1586 bp in the SK2-S promoter. An increase in SK2-S promoter activity was observed in PC12 cells transiently transfected with a wild-type SK2-S promoter-luciferase reporter gene construct and treated with aldosterone or dexamethasone. The mineralocorticoid receptor (MR) antagonist spironolactone or the glucocorticoid receptor (GR) antagonist mifepristone fully inhibited aldosterone or dexamethasone activation of the SK2-S promoter, respectively. SK2-S promoter activity was also induced by the cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide). Antisense oligonucleotides directed to NF-kappaB p65 or p50 suppressed SK2-S transcription induced by C2-ceramide. Deletion studies showed that only the -1586 bp NF-kappaB binding site was necessary for maximum C2-ceramide response. Finally, we showed that activation of GRs but not of MRs repressed the NF-kappaB-mediated induction of SK2-S transcription. These findings suggest a possible transcriptional cross talk between GRs and NF-kappaB in the intronic promoter regulation of SK2-S channel gene transcription.

Adrenomedullin Inhibits Adipogenesis Under Transcriptional Control of Insulin

We generated preadipocyte cell lines impaired in adrenomedullin production through integration of an adrenomedullin small interfering RNA expression vector. The reduction of adrenomedullin synthesis strongly accelerated adipose differentiation. These results were bolstered when overexpression of active adrenomedullin peptide led to delayed differentiation. Therefore, we propose that adrenomedullin is an antiadipogenic factor. Moreover, we checked whether insulin, a proadipogenic factor, regulates expression of adrenomedullin. We observed that insulin had an inhibitory effect on adrenomedullin expression in isolated human adipocyte cells. This response was dose dependent and was reversed by resistin, a new anti-insulin agent. We quantified circulating adrenomedullin in healthy obese patients and observed a threefold increase of adrenomedullin compared with lean patients. Furthermore, adrenomedullin plasma levels are negatively correlated to plasma insulin levels in these obese patients. The insulin inhibitory response was also observed in vivo in Sprague-Dawley rats but not in the insulin-resistant Zucker rat, suggesting that adrenomedullin expression is upregulated in insulin-resistant adipose cells. Using adrenomedullin promoter-luciferase reporter gene constructs, we have shown that the adrenomedullin response to insulin is mediated by insulin-responsive elements. These findings provide new insight into fat mass development and the relationship between obesity and elevated circulating adrenomedullin levels in diabetic patients.

Functional analysis of the polymorphism − 211C>T in the regulatory region of the human ABCC3 gene

The multidrug resistance protein 3 (MRP3/gene symbol: ABCC3) is an ATP-dependent efflux pump mediating the transport of endogenous glucuronides and conjugated drug metabolites across cell membranes. In humans the hepatic expression of ABCC3 mRNA seems to be influenced by the polymorphism C>T at the position − 211 in the promoter of the ABCC3 gene. The aim of this study was to investigate the possible mechanisms of how this SNP influences the MRP3 expression. Promoter luciferase reporter gene constructs representing 0.5, 1.1, 4.4, and 8.1 kb upstream of the translational start site were cloned with cytosine or thymine at position − 211 and transfected into HepG2, Caco-2, and LS174T cells. Reporter gene activity was dependent on the length of the promoter sequence but interestingly not on the nucleotide at position − 211. Cotransfection with FTF cDNA (Fetoprotein Transcription Factor) binding to elements near the − 211 polymorphism increased promoter activity in all constructs except the 0.5 kb fragment also independently of the − 211 SNP. Taken together, we did not find any influence of the − 211C>T ABCC3 promoter polymorphism on either the basal or the FTF induced reporter gene activity. Whether other tissue specific mechanisms reveal an impact of this SNP on the in vivo regulation of MRP3 remains to be determined.

Nerve Growth Factor Regulates Adrenergic Expression

The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC-12 cells transfected with a rat phenylethanolamine N-methyl-transferase (PNMT) promoter-luciferase reporter gene construct pGL3RP893. NGF treatment increased PNMT promoter-driven luciferase activity in a dose- and time-dependent manner. Induction was attenuated by inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway ( approximately 60%) but not by inhibition of the protein kinase A (PKA), protein kinase C, phosphoinositol kinase, or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF-responsive sequences lay within the proximal -392 base pairs (bp) of PNMT promoter, wherein binding elements for Egr-1 (-165 bp) and Sp1 (-48 bp) reside. Western analysis further showed that NGF increased nuclear levels of Egr-1, but not Sp1 or the catalytic subunit of PKA. Gel mobility shift assays showed increased potential for Egr-1, but not Sp1, protein-DNA binding complex formation. Mutation of either the Egr-1 or Sp1 binding sites in the PNMT promoter attenuated NGF activation. NGF, combined with pituitary adenylyl cyclase-activating protein (PACAP), another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control seems to be another important mechanism by which neurotrophins regulate the adrenergic phenotype. NGF, PACAP, and a combination of the two stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein, but to different extents.

Activation of the rat scavenger receptor class B type I gene by PPARα

Peroxisomal proliferator activated receptor α (PPARα) is activated by fibrate drugs which are known to protect against atherosclerosis. The present study examines the effects of PPARα on SR-BI expression. For this study, a rat SR-BI promoter-luciferase reporter gene construct was co-transfected into different cell lines with expression vectors that encode for PPARα ± retinoic X receptor α (RXRα). PPARα/RXR increased the activity of the SR-BI promoter, an effect that was enhanced by clofibrate. Sequence analysis of the rat SR-BI promoter revealed the presence of a putative peroxisomal proliferator response element (PPRE) at bp −1622. Electrophoretic mobility shift assays demonstrated that PPARα and RXRα are able to bind to the SR-BI PPRE motif. In addition, mutational analysis studies confirmed that this PPRE motif is responsible for the PPARα/RXRα-dependent activation of the rat SR-BI promoter in the cell lines examined.

CREB-AP1 Protein Complexes Regulate Transcription of the Collagen XXIV Gene (Col24a1) in Osteoblasts

Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which displays unique structural features of invertebrate fibrillar collagens and is expressed predominantly in bone tissue. Here we report the characterization of the proximal promoter of the mouse gene (Col24a1) and its regulation in osteoblastic cells. Using well characterized murine models of osteoblast differentiation, we found that the Col24a1 gene is activated sometime before onset of the late differentiation marker osteocalcin. Additional analyses revealed that Col24a1 produces equal amounts of two alternatively spliced products with different 5'-untranslated sequences that originate from distinct transcriptional start sites. Cell transfection experiments in combination with DNA binding assays demonstrated that Col24a1 promoter activity in ROS17/2.8 osteosarcoma cells is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1, and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2, or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24a1 promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Col24a1 gene. Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB-AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.

Chronic nicotine effects on adrenergic function

Smokers suggest that smoking relaxes them but after smoking, the two stress hormones epinephrine (EPI) and cortisol rapidly rise. Smoking is highly associated with cardiovascular pathology and morbidity, and EPI, a major vasopressor, may be an underlying factor. We propose that nicotine (NIC), the major addictive component in cigarettes, acts directly via nicotinic receptors and indirectly via the hypothalamic-pituitary-adrenal axis to elevate EPI biosynthesis by activating the EPI-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). In PC12 cells transfected with a PNMT promoter-luciferase reporter gene construct, NIC (100 μM) stimulates luciferase activity in a dose- and time-dependent manner, with activity highest at 6 hr (2.5-fold), back to basal levels by 24 hr and increasing again with continued exposure. In combination with DEX (1 μM), synergistic activation is apparent at 24 hr. In addition, NIC and DEX induce endogenous PNMT mRNA and protein in untransfected PC12 cells. Whether the combination leads to cooperative stimulation remains to be determined. When NIC is administered chronically to rats (1.6 mg/kg i.p. twice daily for 7 days), PNMT mRNA and protein rise 2.0 to 2.5-fold in the adrenal medulla. Finally, in both NIC-treated PC12 cells (24 hr) or adrenal medulla of NIC-treated rats (7 days), mRNA for the immediate early gene transcription factor Egr-1 markedly rises. Together these results suggest that NIC and glucocorticoids, while independently activating the PNMT gene, may synergistically stimulate PNMT with chronic exposure, Egr-1 being one factor mediating induction.

DETECTION OF ANDROGENIC ACTIVITY IN EMISSIONS FROM DIESEL FUEL AND BIOMASS COMBUSTION

The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor-mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the samples were used as tools to separate active from inactive fractions, with the goal of identifying the specific compounds responsible for the activity. A significant androgenic response was detected from the diesel emission. High-performance liquid chromatographic fractionation of the sample indicated that significant androgenic activity was retained in three fractions. 4-Hydroxybiphenyl was identified from the most active fraction using gas chromatography/mass spectroscopy. This purified compound was then tested at doses from 1 nM to 100 microM. 4-Hydroxybiphenol exhibited antagonist activity at low concentrations and agonist activity at high concentrations. A competitive-binding assay confirmed binding to the androgen receptor, with a median inhibitory concentration for radioligand binding of approximately 370 nM. Significant androgenic activity also was detected in the wood burn samples, but we were unable to identify the specific chemicals responsible for this endocrine activity. The present study demonstrates that in vitro bioassays can serve as sensitive bioanalytical tools to aid in characterization of complex environmental mixtures.