Response Element-mediated Transcription Research Articles

GR-mediated anti-inflammation of α-boswellic acid: Insights from in vitro and in silico studies

Although multiple bioactivities of α-boswellic acid have been reported, the molecular mechanism of its anti-inflammatory action is not yet clear. Hence, glucocorticoid receptor (GR)-mediated anti-inflammation of α-boswellic acid was investigated in this work. Fluorescence polarization assay suggested that α-boswellic acid bound to GR with IC50 value of 658.00 ± 0.21 μM. Upon binding to α-boswellic acid, GR translocated from cytoplasm into nucleus of HeLa cells, facilitating sequential transcriptional regulation of GR-related genes. Luciferase reporter assay suggested that α-boswellic acid lacked GR transcriptional activity, indicating its potential as a dissociative GR ligand. Interestingly, α-boswellic acid selectively modulated the anti-inflammatory gene CBG (marker for GR transrepression), while leaving the “side-effect” gene TAT (marker for GR transactivation) unaffected in HepG2 cells. Furthermore, α-boswellic acid inhibited lipopolysaccharide-stimulated cytokines production in U937 macrophages, confirming its anti-inflammation property in vitro. Molecular docking showed that both hydrogen-bonding and hydrophobic interactions helped to stabilize α-boswellic acid-GR binding. Their binding stability was further confirmed in a 70-ns dynamics simulation. In summary, α-boswellic acid could bind to and translocate GR but did not induce glucocorticoid response element-mediated transcription. Since α-boswellic acid showed the dissociated characteristic that separated transrepression from transactivation, it might be a selective GR modulator against inflammatory disorders.

Isolation of Adenosine and Cordysinin B from Anredera cordifolia that Stimulates CRE-Mediated Transcription in PC12 Cells

AbstractAlzheimer’s disease is a typical neurodegenerative disorder, and its prevention or treatment poses great concern in advanced countries. In our survey of numerous natural resources with neurotrophic activities, we found that Anredera cordifolia improved memory impairment and increased cyclic adenosine monophosphate (AMP) response element-mediated transcription, an important step in signal transduction for memory formation. The extracts of this food were dissolved in methanol and then partitioned with three organic solvents and water, separating into n-hexane, ethyl acetate, n-butanol, and water layers. The n-butanol layer with the strongest activity on cyclic AMP-response element-dependent transcription was fractionated using silica gel column chromatography and then the activity was monitored using preparative high-performance liquid chromatography to give adenosine and cordysinin B, respectively. Both compounds showed a concentration-dependent increase in cyclic AMP-response element-mediated transcription activity. These results suggest that both adenosine and cordysinin B may participate in improving the action of A. cordifolia on memory impairment, and these actions, at least in part, result from the activation of adenosine A1, A2A, and A2B receptors.

Calcineurin controls gene transcription following stimulation of a Gαq-coupled designer receptor

Stimulation of Gaq-coupled receptors triggers the activation of gene transcription via a rise of intracellular Ca2+. To investigate the role of the Ca2+/calmodulin-dependent phosphatase calcineurin in regulating transcription following Gαq-coupled receptor stimulation, we used a gain-of-function approach and expressed ΔCnA, a constitutively active mutant of calcineurin A. Furthermore, we expressed hM3Dq, a designer receptor that is specifically coupled to Gαq and can be activated by the pharmacological compound clozapine-N-oxide. Stimulation of hM3Dq or expression of ΔCnA induced transcription of a reporter gene controlled by the calcineurin substrate nuclear factor of activated T cells (NFAT), suggesting that calcineurin increased NFAT-regulated gene transcription. In contrast, expression of ΔCnA attenuated hM3Dq-induced biosynthesis of the transcription factors c-Fos and Egr-1 and reduced both c-Fos and Egr-1 promoter activities. A dissection of the c-Fos and Egr-1 promoters revealed that calcineurin inhibited serum response element-mediated transcription. In particular, the expression of ΔCnA reduced the transcriptional activity of the ternary complex factor Elk-1 following stimulation of hM3Dq receptors. Furthermore, ΔCnA reduced the transcriptional activity of the transcription factor CREB and thus attenuated transcription mediated by the cAMP response element. In summary, we show that calcineurin functions as a positive and negative modulator of gene transcription.

Stimulation of TRPM3 channels increases the transcriptional activation potential of Elk-1 involving cytosolic Ca2+, extracellular signal-regulated protein kinase, and calcineurin

Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in “BPA-free” products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10−11–10−4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer.

Specificity of Stress‐Responsive Transcription Factors Nrf2, ATF4, and AP‐1

Cellular stress leads to an upregulation of gene transcription. We asked if there is a specificity in the activation of the stress-responsive transcription factors Nrf2, ATF4, and AP-1/c-Jun, or if activation of these proteins is a redundant cellular answer toward extracellular stressors. Here, we show that oxidative stress, induced by stimulation of the cells with the oxidant arsenite, strongly activated gene transcription via the stress-responsive element (StRE), while phorbol ester or tunicamycin, activators of AP-1/c-Jun or ATF4, respectively, activated AP-1 or nutrient-sensing response element-mediated transcription. Preincubation of the cells with N-acetyl-cysteine or overexpression of thioredoxin selectively attenuated arsenite-induced upregulation of StRE-regulated transcription. Expression of either dominant-negative or constitutively active mutants of Nrf2, ATF4, or c-Jun confirmed that distinct transcription units are regulated by these transcription factors. Physiological stimuli involving the activation of either Gαq-coupled designer receptors or the protein kinases c-Jun N-terminal protein kinase or p38 strongly stimulated transcription via AP-1/c-Jun, with minimal effects on Nrf2 or ATF4-responsive promoters. Thus, activation of transcription by extracellular signaling molecules shows specificity at the level of the chemical nature of the signaling molecule, at the level of the intracellular transduction process, and at the level of signal-responsive transcription factors. J. Cell. Biochem. 118: 127-140, 2017. © 2016 Wiley Periodicals, Inc.

Photic Phase-Response Curve in 2 Strains of Mice with Impaired Responsiveness to Estrogens

Steroid hormones including estrogens modulate the expression of daily activity and circadian rhythms, including free-running period, phase angle of activity onset, and response to light. The mechanisms underlying these effects, however, are not fully understood. We tested the hypothesis that estrogen signaling is required for photic responsiveness of the circadian timing system. We used estrogen receptor subtype 1 (ESR1) knock-out mice (ERKO) and nonclassic estrogen receptor knock-in mice (NERKI). ERKO animals are unable to respond to estrogen at ESR1, and NERKI animals lack the ability to respond to estrogens via estrogen response element-mediated transcription but still respond via nonclassical mechanisms. We analyzed behavioral shifts in activity onset in response to 1-h light pulses given across the subjective 24-h day in gonadally intact male and female NERKI, ERKO, and wild-type (WT) littermates. We also examined Fos protein expression in the suprachiasmatic nucleus, the site of the master circadian pacemaker, at 2 times of day. We found a significant effect of genotype on phase shifts in response to light pulses given in the subjective night. Female WT mice had a significantly larger phase response than ERKO females during the early subjective night (phase shift of 98 min and 58 min, respectively; p < 0.05). NERKI females were intermediate to WT and ERKO females, suggesting a contribution of nonclassical estrogen signaling on circadian timekeeping functions. This genotype effect is not observed in males; they did not have a difference in phase shifts following a light pulse at any time point. WT males, however, shifted an average of 47 min less than did females at zeitgeber time (ZT) 16 (ZT 0 lights-on and ZT 12 lights-off). These data indicate that estrogens modify the response of the circadian timekeeping system to light via classical and nonclassical signaling pathways.

Modification of p115RhoGEF Ser330 regulates its RhoGEF activity

p115RhoGEF is a member of a family of Rho-specific guanine nucleotide exchange factors that also contains a regulator of G protein signaling homology domain (RH-RhoGEFs) that serves as a link between Gα13 signaling and RhoA activation. While the mechanism of regulation of p115RhoGEF by Gα13 is becoming well-known, the role of other regulatory mechanisms, such as post-translational modification or autoinhibition, in mediating p115RhoGEF activity is less well-characterized. Here, putative phosphorylation sites on p115RhoGEF are identified and characterized. Mutation of Ser330 leads to a decrease in serum response element-mediated transcription as well as decreased activation by Gα13 in vitro. Additionally, this study provides the first report of the binding kinetics between full-length p115RhoGEF and RhoA in its various nucleotide states and examines the binding kinetics of phospho-mutant p115RhoGEF to RhoA. These data, together with other recent reports on regulatory mechanisms of p115RhoGEF, suggest that this putative phosphorylation site serves as a means for initiation or relief of autoinhibition of p115RhoGEF, providing further insight into the regulation of its activity.

Isoflavones suppress cyclic adenosine 3′,5′-monophosphate regulatory element-mediated transcription in osteoblastic cell line

Soy isoflavones have been implicated to exert benefit on bone loss in postmenopausal women. Isoflavones can induce estrogen response element-mediated transcription in osteoblastic cells. In the present study, we investigate whether isoflavones genistein and daidzein regulate target gene transcription through cAMP regulatory element (CRE) in osteoblastic cells. It was found that 17β-estradiol (E 2), genistein and daidzein suppressed the transcriptional activity of CRE-luciferase reporter gene in human osteoblastic cell line MG-63 cells. E 2 and genistein but not daidzein inhibited the cAMP analogue 8-Br cAMP-induced transcription of CRE reporter gene. Both genistein and E 2 inhibited basal and cAMP-induced mRNA levels of endogenous estrogen responsive genes containing CRE/CRE-like elements in their promoter regions, including interleukin (IL) 8 and serum- and glucocorticoid-inducible kinase 1 (SGK1). Daidzein inhibited basal and cAMP-induced IL-8, but not SGK1 mRNA expression. The inhibitory effects of E 2, genistein and daidzein on CRE-mediated transcription activity were enhanced by estrogen receptor (ER) α overexpression in MG-63 cells, which could be blocked by nonselective ER antagonists ICI182780, 4-OH tamoxifen and specific ERα antagonist MPP. Genistein and daidzein, but not E 2 treatment, caused a significant decrease in CRE-mediated transcription activity in ERβ-transfected MG-63 cells, which could be blocked by ICI182780, 4-OH tamoxifen and the selective ERβ antagonist ( R, R)-5,11-diethyl-5.6,11,12-tetradro-2,8-chrysenediol. Our results indicate that isoflavones genistein and daidzein might modulate bone remodeling through ERs by regulating target gene expression through the CRE motifs.

Influence of selective estrogen receptor modulators on interleukin-6 expression in human retinal pigment epithelial cells (ARPE-19)

Since estrogen and selective estrogen receptor modulators can inhibit inflammatory responses, we studied the regulatory role of several selective estrogen receptor modulators on interleukin-6 (IL-6) expression in human retinal pigment epithelial cells (ARPE-19). ARPE-19 cells were exposed to lipopolysaccharide with simultaneous exposure to different selective estrogen receptor modulators with the secretion of IL-6 cytokine being analyzed by enzyme-linked immunosorbent assay (ELISA). We demonstrate that 17β-estradiol and HM-D, a novel selective estrogen receptor modulator compound, clearly reduced the IL-6 expression levels after lipopolysaccharide exposure in ARPE-19 cells. Molecular effects of selective estrogen receptor modulators and estrogen on the estrogen response element-mediated transcription were studied using MCF-7 and ARPE-19 cell lines carrying the estrogen response element-luciferase reporter gene. Estrogen and HM-D stimulated the activity of estrogen response element-reporter gene in MCF-7 cells but did not affect the activity in ARPE-19 cells. In addition, HM-D did not activate estrogen receptor α when studied by nuclear receptor peptide estrogen receptor α ELISA in ARPE-19 cells. These results indicate that estrogen and HM-D can suppress the lipopolysaccharide-induced inflammatory response but signalling is not mediated through estrogen response element transcription in human retinal pigment epithelial cells.

The Constitutively Active Orphan G-protein-coupled Receptor GPR39 Protects from Cell Death by Increasing Secretion of Pigment Epithelium-derived Growth Factor

GPR39 is a constitutively active orphan G-protein-coupled receptor capable of increasing serum response element-mediated transcription. We found GPR39 to be up-regulated in a hippocampal cell line resistant against diverse stimulators of cell death and show that its overexpression protects against oxidative and endoplasmic reticulum stress, as well as against direct activation of the caspase cascade by Bax overexpression. In contrast, silencing GPR39 rendered cells more susceptible to cell death. An array analysis of transcripts induced by GPR39 revealed up-regulation of RGS16 (inhibitor of G-protein signaling 16), which suggested coupling to Galpha(13) and induction of serum response element-mediated transcription by the small GTPase RhoA. In line with this, co-expression of GPR39 with RGS16, dominant-negative RhoA, or serum response factor abolished cell protection, whereas overexpression of the serum response factor protected from cell death. Further downstream the signaling cascade, GPR39 overexpression leads to increased secretion of the cytoprotective pigment epithelium-derived growth factor (PEDF). Medium conditioned by cells overexpressing GPR39 contained 4-fold more PEDF, and when stripped off it lost most but not all of its protective properties. We conclude that GPR39 is a novel inhibitor of cell death, which might represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress like cancer, ischemia/reperfusion injury, and neurodegenerative disease.

Tamoxifen Induction of CCAAT Enhancer-binding Protein α Is Required for Tamoxifen-induced Apoptosis

Low concentrations of tamoxifen or its active metabolite 4-hydroxytamoxifen (OHT) induce estrogen receptor alpha (ERalpha)-dependent apoptosis. To analyze the pathway of OHT-ERalpha-induced apoptosis, we developed stably transfected lines of HeLa cells expressing wild-type ER and an inactive mutant ERalpha unable to bind estrogen response elements. HeLa cells expressing the mutant ERalpha and HeLa cells expressing wild-type ERalpha in which the ER was knocked down with an ER-specific small interfering RNA were not killed by Tam or OHT, suggesting that estrogen response element-mediated transcription is required for Tam- and OHT-induced apoptosis. Microarray analysis to identify a gene(s) whose expression is important in OHT-ER-mediated apoptosis identified 19 mRNAs that OHT up-regulated by >1.6-fold and 15 down-regulated mRNAs. Gene function and the time course of induction by OHT-ERalpha led us to further investigate CCAAT enhancer-binding protein alpha (C/EBPalpha), which has roles in cell cycle progression and apoptosis, and p21. Quantitative reverse transcription-PCR, Western blot analysis, and RNA interference knockdown suggest that cell cycle arrest resulting from OHT-ERalpha induction of p21 may facilitate apoptosis. OHT-ERalpha, but not E2-ERalpha, induced C/EBPalpha mRNA and protein. RNA interference knockdown of C/EBPalpha nearly abolished OHT-ERalpha-induced apoptosis. We isolated stable cell lines that were resistant to OHT-induced apoptosis, contain full-length functional ERalpha, and undergo apoptosis in response to etoposide. In these OHT-resistant cell lines both before and after OHT treatment, C/EBPalpha levels are much lower than in OHT-sensitive cells. These studies establish a novel molecular site responsible for Tam- and OHT-ERalpha-induced apoptosis of cancer cells.

Discovery of NM23-H2 as an estrogen receptor β-associated protein: Role in estrogen-induced gene transcription and cell migration

The regulation of the estrogenic responses may be influenced by the proteins that associate with estrogen receptors (ERs) rather than solely with the receptors themselves. ERβ is expressed in blood vessels and may play an important role in vascular disease. We hypothesized that specific proteins interact with ERβ to modulate its response to estrogens. By means of a yeast two hybrid screen, we discovered that NM23-H2, a multi-faceted protein associates specifically with ERβ. NM23-H2 and ERβ consistently co-localize in a variety of human tissues (e.g. breast tissue), whereas ERα and NM23-H2 did not co-localize. Estrogen response element-mediated transcription increased by 97% when NM23-H2 and ERβ were over-expressed in MCF-7 cells ( p ≤ 0.001). Moreover, there was a synergistic effect of NM23-H2 over-expression with estrogen treatment on the reduction of MCF-7 cell migration ( p ≤ 0.001). These results suggest that NM23-H2 associates with ERβ and is capable of modulating estrogen-induced gene transcription, as well as cell migration. Hence, NM23-H2 may play an important role in modulating the response to endogenous and exogenous estrogens, perhaps even within the context of vascular disease.

Induction of Osteoblast Differentiation by Selective Activation of Kinase-Mediated Actions of the Estrogen Receptor

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Estradiol Reduces Nonclassical Transcription at Cyclic Adenosine 3′,5′-Monophosphate Response Elements in Glioma Cells Expressing Estrogen Receptor Alpha

Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17beta-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) alpha (C6ERalpha) or rat ERbeta (C6ERbeta). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ERalpha, and C6ERbeta cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ERalpha but had no effect on reporter gene expression in C6ERbeta or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ERalpha and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.

Nongenotropic, Anti-Apoptotic Signaling of 1α,25(OH)2-Vitamin D3 and Analogs through the Ligand Binding Domain of the Vitamin D Receptor in Osteoblasts and Osteocytes: MEDIATION BY Src, PHOSPHATIDYLINOSITOL 3-, AND JNK KINASES

Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1alpha,25(OH)2-vitamin D3 (1alpha,25(OH)2D3), and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [3H]1alpha,25(OH)2D3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1alpha,25(OH)2D3 and the 6-s-cis-locked 1alpha,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1alpha,25(OH)2D3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1alpha,25(OH)2D3, indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1alpha,25(OH)2D3.

Beta-amyloid peptide at sublethal concentrations downregulates brain-derived neurotrophic factor functions in cultured cortical neurons.

The accumulation of beta-amyloid (Abeta) is one of the etiological factors in Alzheimer's disease (AD). It has been assumed that the underlying mechanism involves a critical role of Abeta-induced neurodegeneration. However, low levels of Abeta, such as will accumulate during the course of the disease, may interfere with neuronal function via mechanisms other than those involving neurodegeneration. We have been testing, therefore, the hypothesis that Abeta at levels insufficient to cause degeneration (sublethal) may interfere with critical signal transduction processes. In cultured cortical neurons Abeta at sublethal concentrations interferes with the brain-derived neurotrophic factor (BDNF)-induced activation of the Ras-mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. The effect of sublethal Abeta(1-42) on BDNF signaling results in the suppression of the activation of critical transcription factor cAMP response element-binding protein and Elk-1 and cAMP response element-mediated and serum response element-mediated transcription. The site of interference with the Ras/ERK and PI3-K/Akt signaling is downstream of the TrkB receptor and involves docking proteins insulin receptor substrate-1 and Shc, which convey receptor activation to the downstream effectors. The functional consequences of Abeta interference with signaling are robust, causing increased vulnerability of neurons, abrogating BDNF protection against DNA damage- and trophic deprivation-induced apoptosis. These new findings suggest that Abeta engenders a dysfunctional encoding state in neurons and may initiate and/or contribute to cognitive deficit at an early stage of AD before or along with neuronal degeneration.

Protein Kinase A Activation of Estrogen Receptor α Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation

17beta-Estradiol (E2)-stimulated estrogen receptor (ERalpha) transcription is accompanied by protein degradation via the 26S-proteasome pathway. Inhibition of proteasome activity stabilizes ERalpha protein and abolishes E2-activated transcription, suggesting functional linkages between transcription and degradation. It is not known whether ligand-independent ERalpha activation is coupled to proteolysis. In pituitary cells, forskolin (FSK) stimulates ERalpha transcription through the protein kinase A (PKA) pathway. This study examined interactions between E2-dependent and PKA-stimulated pathways in GH(3) cells by measuring transcription of a transfected reporter gene and endogenous ERalpha levels. E2 stimulated estrogen response element-mediated transcription 2- to 3-fold and decreased ERalpha protein levels to 40%. In contrast, FSK stimulated ERalpha transcription without decreasing ERalpha protein. Treatment with FSK plus E2 resulted in synergistic ERalpha transactivation, and FSK specifically prevented E2-induced ERalpha degradation. PKA is required for protection and was prevented by H89 (a PKA inhibitor), but not PD98059 (a MAPK kinase inhibitor). Propyl-pyrazole-triol and R,R-diethyl-tetrahydrochrysene, selective ERalpha agonists, reduced ERalpha protein by 50% while stimulating ERalpha transcriptional activity 4- to 8-fold. The antagonist ICI 182,780 similarly decreased ERalpha levels, but prevented ER activation. FSK prevented all ligand-induced ERalpha degradation. Lactacystin, a proteasome inhibitor, abolished E2-stimulated, but not FSK-stimulated, ERalpha transcription. Thus, stimulation of ERalpha transcription by the PKA-dependent pathway is dissociated from receptor degradation and proteasome activity. These data suggest a mechanism of ERalpha transcriptional activation by PKA that is distinct from E2 activation and that may contribute to the synergistic transcriptional activation of ERalpha by ligand-dependent and PKA-dependent pathways.

Decreased cAMP Response Element-mediated Transcription: AN EARLY EVENT IN EXON 1 AND FULL-LENGTH CELL MODELS OF HUNTINGTON′S DISEASE THAT CONTRIBUTES TO POLYGLUTAMINE PATHOGENESIS

Huntington's disease (HD) is one of nine neurodegenerative diseases caused by an expanded polyglutamine (polyQ) tract within the disease protein. To characterize pathways induced early in HD, we have developed stable inducible PC12 cell lines expressing wild-type or mutant forms of huntingtin exon 1 fragments or the full-length huntingtin protein. Three cAMP response element-binding protein (CREB)-binding protein-dependent transcriptional pathways, regulated by cAMP response element (CRE), retinoic acid response element, and nuclear factor kappaB, show abnormalities in our exon 1 cell model. Of these, the CRE pathway shows the earliest disruption and is significantly down-regulated as early as 12 h following mutant htt transgene induction. This pathway is also the only one of the three that is similarly perturbed in our full-length HD model, where it is also down-regulated at an early time point, compatible with observations in HD brains. Reduced CRE-dependent transcription may contribute to polyQ disease pathogenesis because overexpression of transcriptionally active CREB, but not an inactive form of the protein, is able to protect against polyQ-induced cell death and reduce aggregation.