Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.

Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.

vgf is a neurotrophin response-specific, developmentally regulated gene that codes for a neurosecretory polypeptide. Its transcription in neuronal cells is selectively activated by the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3, which induce survival and differentiation, and not by epidermal growth factor. We studied a short region of the rat vgf promoter which is essential for its regulated expression. A cyclic AMP response element (CRE) within this region is necessary for NGF induction of vgf transcription. Two sites upstream of CRE, an E box and a CCAAT sequence, bind nuclear protein complexes and are involved in transcriptional control. The E box has a dual role. It acts as an inhibitor in NIH 3T3 fibroblasts, together with a second E box located downstream, and as a stimulator in the NGF-responsive cell line PC12. By expression screening, we have isolated the cDNA for a basic helix-loop-helix transcription factor, a homolog of the HTF4/HEB E protein, that specifically binds the vgf promoter E box. The E protein was present in various cell lines, including PC12 cells, and was a component of a multiprotein nuclear complex that binds the promoter in vitro. The E box and CRE cooperate in binding to this complex, which may be an important determinant for neural cell-specific expression.

Nuclear factor-kappaB and cAMP response element binding protein mediate opposite transcriptional effects on the Flk-1/KDR gene promoter.

-The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues. Despite its biological relevance, little is known about the molecular mechanisms controlling its expression. In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-kappaB (NF-kappaB)-related antigens bind specific sequences in the Flk-1/KDR promoter. Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-alpha, an activator of NF-kappaB, stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-kappaB, increase Flk-1/KDR promoter activity 10- to 20-fold. Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element. In contrast, specific mutations in the NF-kappaB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression. These results suggest that opposing signaling pathways, activating NF-kappaB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity.

UltraRapid communication : nuclear factor-kappaB and cAMP response element binding protein mediate opposite transcriptional effects on the flk-1/KDR gene promoter

-The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues. Despite its biological relevance, little is known about the molecular mechanisms controlling its expression. In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-kappaB (NF-kappaB)-related antigens bind specific sequences in the Flk-1/KDR promoter. Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-alpha, an activator of NF-kappaB, stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-kappaB, increase Flk-1/KDR promoter activity 10- to 20-fold. Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element. In contrast, specific mutations in the NF-kappaB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression. These results suggest that opposing signaling pathways, activating NF-kappaB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity. texfThe full text of this article is available at http://www.circresaha.org. Key Words: vascular endothelial growth factor receptor promoter nuclear factor-kappaB transcription angiogenesis Web Site Feature

Retinoids induce fibroblast growth factor-2 production in endothelial cells via retinoic acid receptor alpha activation and stimulate angiogenesis in vitro and in vivo.

The effect of retinoic acid (RA) on endothelial cells is still controversial and was examined in the present study. In bovine aortic endothelial cells (BAECs), all-trans RA (ATRA) and 9-cis RA (9CRA), but not 13-cis RA (13CRA), induced fibroblast growth factor-2 (FGF-2) production and exhibited a biphasic dose-dependent effect to enhance BAEC proliferation and differentiation into tubular structures on reconstituted basement membrane proteins (Matrigel); both processes were inhibited by FGF-2-neutralizing antibody. The pan RA receptor (RAR)-selective ligand (E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl)-1-propenyl] benzoic acid and the RARalpha-selective ligand 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid stimulated the production of FGF-2, whereas the addition of the RARalpha-antagonist RO 41-5253 inhibited this effect. In BAECs, the forced expression of RARalpha, but not RARbeta or RARgamma, enhanced FGF-2 production, whereas the RARalpha-dominant negative, Delta403, blocked this effect. Furthermore, RARalpha overexpression directly stimulated BAEC differentiation on Matrigel and potentiated the effects of ATRA in this assay. Finally, ATRA-treated BAECs coinjected with Matrigel subcutaneously in mice induced neovascularization within the Matrigel plug, and ATRA also enhanced angiogenesis in the chicken chorioallantoic membrane assay. In conclusion, RA can stimulate endothelial cell proliferation and differentiation in vitro via enhanced RARalpha-dependent FGF-2 production, and it can also induce angiogenesis in vivo. The full text of this article is available at http://www.circresaha.org.

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression.

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

Analysis of the Myc and Max interaction specificity with lambda repressor-HLH domain fusions.

The basic helix-loop-helix domain (bHLH) is present in a large class of transcriptional regulators involved in developmental processes and oncogenesis. It determines DNA binding and specific homo- and heterodimeric protein associations, crucial for protein function. Myc and Max belong to a subset of HLH proteins, containing a leucine zipper (LZ) adjacent to the bHLH domain. They differ in dimerization and functional properties such as DNA binding and transcriptional activation, and their association is required for malignant transformation by Myc. To analyze the interaction specificity of Myc and Max bHLH-LZ domains, we developed a simple Escherichia coli genetic system, which uses the amino-terminal lambda phage cI repressor as a reporter for dimerization and allows an easy detection of dimeric interactions. By reciprocal exchanges of different Myc and Max subdomains (helix 1, helix 2 and leucine zipper), we showed that the recognition specificity of Max homodimers as well as of Myc/Max heterodimers is entirely determined by the helix 2-leucine zipper region, the major role being played by the leucine zipper. The Myc LZ was found to prevent homodimeric interactions, thus explaining Myc inability to homodimerize efficiently. Moreover, we showed that the system is valid as well for reproducing the interaction of HLH proteins not containing a leucine zipper and that the chimerical proteins maintain sequence-specific DNA binding.

Transcriptionally active drugs improve adenovirus vector performance in vitro and in vivo.

Cytomegalovirus (CMV) promoter is often present in recombinant adenovirus vectors (AdVs) suitable for gene therapy, ensuring high levels of transgene production in a wide range of hosts. Despite this characteristic, the presence of the AdV genome in target cells and tissues typically lasts longer than transgene production that may be rapidly extincted by ill-defined silencing mechanisms. In the present article, it is reported that transcriptionally active drugs, retinoic acid (RA) and histone deacetylase inhibitor trichostatin A (TSA), enhance AdV transgene expression in infected cells and tissues. The association of RA and TSA increased more than seven-fold above control the activity of AdVs encoding for LacZ or VEGF165. This effect was, at least in part, mediated by the direct activation of retinoic acid receptors. Finally, administration of RA and TSA alone at days 0 and 5 after infection prolonged transgene production up to 21 days after infection versus 6-8 days in untreated controls. These results indicate that transcriptionally active drugs improve AdV function and may represent a novel strategy to more efficiently design AdVs for gene therapy interventions.