A binding site for nuclear receptors is required for the differential expression of the aldolase A fast-twitch muscle promoter in body and head muscles

In hind limb muscles, the aldolase A muscle-specific promoter is specifically expressed in glycolytic fast-twitch fibers. Here, we show that in addition, it is expressed at higher levels in trunk and limb muscles than in neck and head muscles independent of their fiber-type content. We have identified by analysis of transgenic mice a DNA element that is required for this differential expression and, to a lesser extent, for fiber-type specificity. We show that members of the nuclear receptor superfamily bind this element in skeletal muscle nuclear extracts. Interestingly, in gel mobility shift assays, different complexes were formed with this sequence in tongue nuclear extracts compared with limb or trunk muscle nuclear extracts. Therefore, binding of distinct nuclear receptors to a single regulatory sequence appears to be associated with the location-dependent expression of the aldolase A muscle-specific promoter.     

Asymptomatic, unruptured carotid-ophthalmic artery aneurysms: angiographical differentiation of each type, operative results, and indications

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.     

Serum response factor mRNA induction in the hypertrophying chicken patagialis muscle

Gene expression in the stretched chicken patagialis (Pat) muscle has not been extensively examined. This study's purpose was to determine the Pat muscle's expression pattern of serum response factor (SRF), skeletal alpha-actin, and MyoD mRNAs after 3 days (onset of stretch), 6 days (end of first week of rapid growth), and 14 days (slowed rate of stretch-induced growth) of stretch. SRF mRNA demonstrated two species (B1 and B2), with B2 being more prevalent in the predominantly fast-twitch Pat muscle, compared with the slow-tonic muscle. Stretch overload increased B1 and B2 SRF mRNA concentrations, and the increase in B1 SRF mRNA concentration was greater at day 6 compared with days 3 or 14. MyoD mRNA concentration was greater in 3-day-stretched Pat muscles, compared with days 6 or 14. Skeletal alpha-actin mRNA concentration was not changed during the study. Gel mobility shift assays demonstrated that SRF binding with serum response element 1 of the skeletal alpha-actin promoter had no altered binding patterns from 6-day-stretched Pat nuclear extracts. It appears that SRF and MyoD mRNAs are induced in the stretch-overloaded Pat muscle but at different time points.     

A novel A/T-rich element mediates ANF gene expression during cardiac myocyte hypertrophy

The induction of the atrial natriuretic factor (ANF) gene during alpha 1-adrenergic stimulation of neonatal rat ventricular myocytes has served as a model for gene expression during cardiac muscle cell hypertrophy. This study describes and identifies a single regulatory element that mediates expression of the ANF gene. Deletional mutations were generated in a 639-bp fragment of the ANF promoter that confers alpha 1-adrenergic inducibility to a luciferase reporter gene in transient transfection assays in ventricular myocytes. The results of gel mobility shift and diethylpyrocarbonate (DEPC) interference studies with nuclear cardiac cell extracts identified the nucleotide contract points for a novel A/T-rich element (ANF-AT) at positions -582/-575 that partially mediates alpha 1-adrenergic inducibility. Mutations in the ANF-AT element reduced alpha-adrenergic inducibility of an ANF-TK-luciferase fusion gene in cardiac cells by 35% but had no effect on expression in other muscle and non-muscle cells tested. Gel mobility supershift assays with antibodies directed against the MEF-2 protein, the homeobox protein MHox, or the zinc finger protein HF-1b, document that these factors are not major components of the endogenous ANF-AT binding activity in cardiac muscle cells. The current study provides evidence for a role for a novel A/T-rich element in the regulation of ANF gene expression in cardiac ventricular myocytes.     

An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells

CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5'-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab')2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.