Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type.

In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (USP) and ecdysone receptor (EcR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Herein we describe Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38), two insect homologues of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. Moreover, transfection experiments in Schneider cells show that DHR38 can affect ecdysone-dependent transcription. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription.

cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Functional domain analysis of glass, a zinc-finger-containing transcription factor in Drosophila.

The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation.

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.

The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death.

Previous studies have implicated the bcl-2 protooncogene as a potential regulator of neuronal survival. However, mice lacking functional bcl-2 exhibited normal development and maintenance of the central nervous system (CNS). Since bcl-2 appears dispensable for neuronal survival, we have examined the expression and function of bcl-x, another member of the bcl-2 family of death regulatory genes. Bcl-2 is expressed in neuronal tissues during embryonic development but is down-regulated in the adult CNS. In contrast, Bcl-xL expression is retained in neurons of the adult CNS. Two different forms of bcl-x mRNA and their corresponding products, Bcl-xL and Bcl-x beta, were expressed in embryonic and adult neurons of the CNS. Microinjection of bcl-xL and bcl-x beta cDNAs into primary sympathetic neurons inhibited their death induced by nerve growth factor withdrawal. Thus, Bcl-x proteins appear to play an important role in the regulation of neuronal survival in the adult CNS.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

No anticorrelation between cyclotron line energy and X-ray flux in 4U 0115+634

We report on an outburst of the high mass X-ray binary 4U 0115+634 with a pulse period of 3.6 s in 2008 March/April as observed with RXTE and INTEGRAL. During the outburst the neutron star’s luminosity varied by a factor of 10 in the 3–50 keV band. In agreement with earlier work we find evidence of five cyclotron resonance scattering features at ~10.7, 21.8, 35.5, 46.7, and 59.7 keV. Previous work had found an anticorrelation between the fundamental cyclotron line energy and the X-ray flux. We show that this apparent anticorrelation is probably due to the unphysical interplay of parameters of the cyclotron line with the continuum models used previously, e.g., the negative and positive exponent power law (NPEX). For this model, we show that cyclotron line modeling erroneously leads to describing part of the exponential cutoff and the continuum variability, and not the cyclotron lines. When the X-ray continuum is modeled with a simple exponentially cutoff power law modified by a Gaussian emission feature around 10 keV, the correlation between the line energy and the flux vanishes, and the line parameters remain virtually constant over the outburst. We therefore conclude that the previously reported anticorrelation is an artifact of the assumptions adopted in the modeling of the continuum.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

The X-ray outburst of the Galactic Centre magnetar SGR J1745−2900 during the first 1.5 year

In 2013 April a new magnetar, SGR 1745−2900, was discovered as it entered an outburst, at only 2.4 arcsec angular distance from the supermassive black hole at the centre of the Milky Way, Sagittarius A*. SGR 1745−2900 has a surface dipolar magnetic field of ∼2 × 1014 G, and it is the neutron star closest to a black hole ever observed. The new source was detected both in the radio and X-ray bands, with a peak X-ray luminosity LX ∼ 5 × 1035 erg s−1. Here we report on the long-term Chandra (25 observations) and XMM–Newton (eight observations) X-ray monitoring campaign of SGR 1745−2900 from the onset of the outburst in 2013 April until 2014 September. This unprecedented data set allows us to refine the timing properties of the source, as well as to study the outburst spectral evolution as a function of time and rotational phase. Our timing analysis confirms the increase in the spin period derivative by a factor of ∼2 around 2013 June, and reveals that a further increase occurred between 2013 October 30 and 2014 February 21. We find that the period derivative changed from 6.6 × 10−12 to 3.3 × 10−11 s s−1 in 1.5 yr. On the other hand, this magnetar shows a slow flux decay compared to other magnetars and a rather inefficient surface cooling. In particular, starquake-induced crustal cooling models alone have difficulty in explaining the high luminosity of the source for the first ∼200 d of its outburst, and additional heating of the star surface from currents flowing in a twisted magnetic bundle is probably playing an important role in the outburst evolution.

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.