Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.

Cloning and characterization of PDE7B, a cAMP-specific phosphodiesterase

A member of the phosphodiesterase (PDE)7 family with high affinity and specificity for cAMP has been identified. Based on sequence homologies, we designate this PDE as PDE7B. The full-length cDNA of PDE7B is 2399 bp, and its ORF sequence predicts a protein of 446 amino acids with a molecular mass of 50.1 kDa. Comparison of the predicted protein sequences of PDE7A and PDE7B reveals an identity of 70% in the catalytic domain. Northern blotting indicates that the mRNA of PDE7B is 5.6 kb. It is most highly expressed in pancreas followed by brain, heart, thyroid, skeletal muscle, eye, ovary, submaxillary gland, epididymus, and liver. Recombinant PDE7B protein expressed in a Baculovirus expression system is specific for cAMP with a Km of 0.03 μM. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an IC50 of 2.1 μM. It is also inhibited by papaverine, dipyridamole, and SCH51866 at higher doses. PDE7A and PDE7B exhibit the same general pattern of inhibitor specificity among the several drugs tested. However, differences in IC50 for some of the drugs suggest that isozyme selective inhibitors can be developed.

Lagging strand replication of rolling-circle plasmids: Specific recognition of the ssoA-type origins in different gram-positive bacteria

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase–ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at –52 and a novel site at –30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at –30 (–30–21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the –30–21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the –30–21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

Golgi Complex Reorganization during Muscle Differentiation: Visualization in Living Cells and Mechanism

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1–2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance

Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39–50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3′ kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34–57%, and association of p85α with both IRS proteins was reduced by 39–52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle.

In addition to the contractile proteins actin and myosin, contractile filaments of striated muscle contain other proteins that are important for regulating the structure and the interaction of the two force-generating proteins. In the thin filaments, troponin and tropomyosin form a Ca-sensitive trigger that activates normal contraction when intracellular Ca is elevated. In the thick filament, there are several myosin-binding proteins whose functions are unclear. Among these is the myosin-binding protein C (MBP-C). The cardiac isoform contains four phosphorylation sites under the control of cAMP and calmodulin-regulated kinases, whereas the skeletal isoform contains only one such site, suggesting that phosphorylation in cardiac muscle has a specific regulatory function. We isolated natural thick filaments from cardiac muscle and, using electron microscopy and optical diffraction, determined the effect of phosphorylation of MBP-C on cross bridges. The thickness of the filaments that had been treated with protein kinase A was increased where cross bridges were present. No change occurred in the central bare zone that is devoid of cross bridges. The intensity of the reflections along the 43-nm layer line, which is primarily due to the helical array of cross bridges, was increased, and the distance of the first peak reflection from the meridian along the 43-nm layer line was decreased. The results indicate that phosphorylation of MBP-C (i) extends the cross bridges from the backbone of the filament and (ii) increases their degree of order and/or alters their orientation. These changes could alter rate constants for attachment to and detachment from the thin filament and thereby modify force production in activated cardiac muscle.