Syncoilin is an intermediate filament protein in activated hepatic stellate cells.

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, ß-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease usually resulting in death of patients by their early twenties. In contrast, mice lacking dystrophin (Dmd(mdx)), appear physically normal despite their underlying muscle pathology. Mice deficient for both dystrophin and the dystrophin-related protein, utrophin, (Dmd(mdx);Utrn-/- mice) die between 6 and 20 weeks of age suffering from severe muscle weakness with joint contractures, pronounced growth retardation and kyphosis, suggesting that dystrophin and utrophin play complementary roles. The exact cause of death in these mice was not determined. Here we show that expression of a truncated utrophin transgene solely within the skeletal muscle of these mutants prevents premature death and the development of any clinical phenotype. In the absence of full-length dystrophin and utrophin, the presence of truncated utrophin also decreases muscle fibre regeneration, relocalizes the dystrophin protein complex to the sarcolemma and re-establishes a normal expression pattern of developmental muscle proteins. These data suggest that Dmd(mdx);Utrn-/- mice succumb to a skeletal muscle defect and that their reduced lifespan is not due to cardiac or neurogenic components. The phenotypic rescue observed demonstrates that the Dmd(mdx);Utrn-/- mice are an ideal model for testing gene delivery protocols for the expression of utrophin or dystrophin in skeletal muscle. To determine the cause of death of the Dmd(mdx):Utrn-/- mice.

The emerging family of dystrophin-related proteins.

Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.

Localization of the DMDL gene-encoded dystrophin-related protein using a panel of nineteen monoclonal antibodies: presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles, and in proliferating brain cell lines.

mAbs have been raised against different epitopes on the protein product of the DMDL gene, which is an autosomal homologue of the X-linked DMD gene for dystrophin. These antibodies provide direct evidence that DMDL protein is localized near acetylcholine receptors at neuromuscular junctions in normal and mdx mouse intercostal muscle. The primary location in tissues other than skeletal muscle is smooth muscle, especially in the vascular system, which may account for the wide tissue distribution previously demonstrated by Western blotting. The DMDL protein was undetectable in the nonjunctional sarcolemma of normal human muscle, but was observed in nonjunctional sarcolemma of Duchenne muscular dystrophy patients, where dystrophin itself is absent or greatly reduced. The expression of DMDL protein is not restricted to smooth and skeletal muscle, however, since relatively large amounts are present in transformed brain cell lines of both glial and Schwann cell origin. This contrasts with the low levels of DMDL protein in adult brain tissue.

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice.

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.

A new DNA marker tightly linked to the fragile X locus (FRAXA).

The fragile X syndrome is the most common cause of familial mental retardation. Genetic counseling and gene isolation are hampered by a lack of DNA markers close to the disease locus. Two somatic cell hybrids that each contain a human X chromosome with a breakpoint close to the fragile X locus have been characterized. A new DNA marker (DXS296) lies between the chromosome breakpoints and is the closest marker to the fragile X locus yet reported. The Hunter syndrome gene, which causes iduronate sulfatase deficiency, is located at the X chromosome breakpoint that is distal to this new marker, thus localizing the Hunter gene distal to the fragile X locus.

UTRN on chromosome 6q24 is mutated in multiple tumors.

Though deletion of the long arm of chromosome 6 is one of the most common aberrations in tumors, its targeted gene(s) has not been convincingly identified. Using a functional screening approach, we found that UTRN (which encodes utrophin, a dystrophin-related protein) at 6q24, when expressed in an antisense orientation, induced cellular transformation, consistent with a tumor suppressor role. Northern blot analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and gene expression arrays all showed that UTRN expression was downregulated in primary tumors compared with matched normal tissues. Several UTRN neighbor genes were not affected in some tumors with UTRN downregulation, suggesting that UTRN was specifically targeted. RT-PCR, coupled with an in vitro transcription and translation assay, revealed inactivation mutations in 21/62 breast cancers, 4/20 neuroblastomas and 4/15 malignant melanomas. Most of the mutations were deletions involving one or more exons that led to the truncation of utrophin. Splicing errors were found in two cases, and nonsense mutation in one case. Overexpression of a wild-type UTRN in breast cancer cells inhibited tumor cell growth in vitro and reduced their tumor potential in nude mice. Our studies suggest that UTRN is a candidate tumor suppressor gene.

Dystrophin and related proteins.

During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.

Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex.

Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function. A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects [1,2]. The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule [3]. The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC [4]. Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem. The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle [5,6]. Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity. These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin. Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin. Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC. This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.

The genetic basis of neuromuscular disorders.

In the last decade, our knowledge of human diseases genes has been growing rapidly as a result of the availability of resources and techniques for mapping and sequencing the human genome. New disease genes are now reported almost weekly. This review illustrates how the identification of genes involved in neuromuscular disorders has led to the characterization of not only novel genes, but also of a variety of different types of genetic mutation. These observations, which include high deletion frequencies, unstable tandem repeat sequences, genomic duplications and triplet repeat expansions, have facilitated the identification of similar types of mutation in other genetic disorders.

Mapping the gene causing X-linked recessive idiopathic hypoparathyroidism to Xq26-Xq27 by linkage studies.

Idiopathic hypoparathyroidism has been reported to occur as an X-linked recessive disorder in two multigeneration kindreds. Affected individuals, who are males, suffer from infantile onset of epilepsy and hypocalcemia, which appears to be due to an isolated congenital defect of parathyroid gland development; females are not affected and are normocalcemic. We have performed linkage studies in these two kindreds (5 affected males, 11 obligate carrier females, and 44 unaffected members) and have used cloned human X chromosome sequences identifying restriction fragment length polymorphisms to localize the mutant gene causing this disorder. Our studies established linkage between the X-linked recessive idiopathic hypoparathyroid gene (HPT) and the DXS98 (4D.8) locus, peak LOD score = 3.82 (theta = 0.05), thereby mapping HPT to the distal long arm of the X chromosome (Xq26-Xq27). Multilocus analysis indicated that HPT is proximal to the DXS98 (4D.8) locus but distal to the F9 (Factor IX) locus, thereby revealing bridging markers for the disease. The results of this study will improve genetic counseling of affected families, and further characterization of this gene locus will open the way for elucidating the factors controlling the development and activity of the parathyroid glands.

Role of beta-dystrobrevin in nonmuscle dystrophin-associated protein complex-like complexes in kidney and liver.

beta-Dystrobrevin is a dystrophin-related and -associated protein that is highly expressed in brain, kidney, and liver. Recent studies with the kidneys of the mdx3Cv mouse, which lacks all dystrophin isoforms, suggest that beta-dystrobrevin, and not the dystrophin isoforms, may be the key component in the assembly of complexes similar to the muscle dystrophin-associated protein complexes (DPC) in nonmuscle tissues. To understand the role of beta-dystrobrevin in the function of nonmuscle tissues, we generated beta-dystrobrevin-deficient (dtnb(-/-)) mice by gene targeting. dtnb(-/-) mice are healthy, fertile, and normal in appearance. No beta-dystrobrevin was detected in these mice by Western blotting or immunocytochemistry. In addition, the levels of several beta-dystrobrevin-interacting proteins, namely Dp71 isoforms and the syntrophins, were greatly reduced from the basal membranes of kidney tubules and liver sinusoids and on Western blots of crude kidney and liver microsomes of beta-dystrobrevin-deficient mice. However, no abnormality was detected in the ultrastructure of membranes of kidney and liver cells or in the renal function of these mice. beta-Dystrobrevin may therefore be an anchor or scaffold for Dp71 and syntrophin isoforms, as well as other associating proteins at the basal membranes of kidney and liver, but is not necessary for the normal function of these mice.

The role of basal and myogenic factors in the transcriptional activation of utrophin promoter A: implications for therapeutic up-regulation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. Utrophin is the autosomal homologue of dystrophin. We previously demonstrated that overexpression of utrophin in the muscles of dystrophin-null transgenic mice completely prevented the phenotype arising from dystrophin deficiency. Two independently regulated promoters control utrophin expression and the upstream promoter (promoter A) is synaptically regulated in muscle. In this study, we have investigated basal regulation and myogenic induction of promoter A. Interactions between Ap2 and Sp1 and their cognate DNA motifs are critical for basal transcription from the minimal promoter region. During differentiation of C2C12 myoblasts in vitro, a 2-fold increase in A-utrophin mRNA level was observed. Expression of a reporter gene, whose transcription was driven by a 1.3 kb promoter A fragment, paralleled expression of the endogenous transcript. Myogenic induction mapped to a conserved upstream muscle-specific E-box, which was shown to bind myogenic regulatory factors, transactivating the promoter up to 18-fold in transient assays. This study provides a basis for further understanding the regulatory mechanisms that control utrophin expression in muscle and may facilitate the development of reagents to effect therapeutic up-regulation of utrophin in DMD.

Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric reductases.

Iron has a variety of functions in cellular organisms ranging from electron transport and DNA synthesis to adenosine triphosphate (ATP) and neurotransmitter synthesis. Failure to regulate the homeostasis of iron can lead to cognition and demyelination disorders when iron levels are deficient, and to neurodegenerative disorders when iron is in excess. In this study we show that three members of the b561 family of predicted ferric reductases, namely mouse cytochrome b561 and mouse and fly stromal cell-derived receptor 2 (SDR2), have ferric reductase activity. Given that a fourth member, duodenal cytochrome b (Dcytb), has previously been shown to be a ferric reductase, it is likely that all remaining members of this family also exhibit this activity. Furthermore, we show that the rat sdr2 message is predominantly expressed in the liver and kidney, with low expression in the duodenum. In hypotransferrinaemic (hpx) mice, sdr2 expression in the liver and kidney is reduced, suggesting that it may be regulated by iron. Moreover, we demonstrate the presence of mouse sdr2 in the choroid plexus and in the ependymal cells lining the four ventricles, through in situ hybridization analysis.

Novel transcribed sequences represented in the complex genomic region 5q13.

YACs from the complex repetitive human genomic region 5q13, spanning the spinal muscular atrophy (SMA) locus, have been searched for transcribed sequences using the method of End Ligation Coincident Sequence Cloning. Six transcripts (PT1-6) have been identified, three of which (PT4, PT5 and PT6) are novel. Five of these elements hybridise to multiple loci in 5q13, but PT5 is single copy and maps very close to markers that show linkage disequilibrium with SMA.

A and B utrophin in human muscle and sarcolemmal A-utrophin associated with tumours.

Utrophin is an autosomal homologue of dystrophin, abnormal expression of which is responsible for X-linked Duchenne and Becker muscular dystrophy. In normal mature muscle utrophin is confined to blood vessels, nerves and myotendinous and neuromuscular junctions. When dystrophin is absent utrophin is abundant on the sarcolemma. This has raised the possibility that up-regulation of utrophin may be of therapeutic benefit. Two full-length transcripts of utrophin, A and B, have been identified, which are regulated by alternatively spliced 5' promoters. In dystrophic mouse muscle, the A isoform is present on the sarcolemma, whereas the B form is confined to blood vessels. We show here using immunohistochemistry and human isoform-specific antibodies that A- and B-utrophin localisation is the same in human muscle. The A isoform is present on the sarcolemma of foetal human muscle fibres, regenerating fibres, fibres deficient in dystrophin and on blood vessels and neuromuscular junctions. B-utrophin is only detected on blood vessels. We also show that muscle adjacent to some soft tissue tumours shows increased sarcolemmal utrophin-A, showing that utrophin and dystrophin can simultaneously localise to the sarcolemma and raising the possibility that factor(s) from the tumour cells or accompanying inflammatory cells may have a role in regulating utrophin.

Cloning and developmental expression analysis of ltd-1, the Caenorhabditis elegans homologue of the mouse kyphoscoliosis (ky) gene.

We have characterized the developmental expression pattern of the Caenorhabditis elegans homologue of the mouse ky gene. The Ky protein has a putative key function in muscle development and has homologues in invertebrates, fungi and a cyanobacterium. The C. elegans Ky homologue gene has been named ltd-1 for LIM and transglutaminase domains gene. The LTD-1::GFP construct is expressed in developing hypodermal cells from the twofold stage embryo through adulthood. These data define the ltd-1 gene as a novel marker for C. elegans epithelial cell development.

Utrophin upregulation in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a devastating, progressive muscle wasting disease for which there is currently no effective treatment. DMD is caused by mutations in the dystrophin gene many of which result in the absence of the large cytoskeletal protein dystrophin at the sarcolemma. Over-expression of utrophin, the autosomal paralogue of dystrophin, as a transgene in the mdx mouse (the mouse model of DMD) has demonstrated that utrophin can prevent the muscle pathology. Thus, up-regulation of utrophin in DMD muscle is a potential therapy for DMD. In this review we discuss recent advances in our understanding of the regulatory pathways controlling utrophin expression and the various approaches that have been applied to increasing the level of utrophin in the mdx mouse. These results are very encouraging and suggest that pharmacological up-regulation of utrophin may well be a feasible approach to therapy for DMD.

Adenovirus-mediated utrophin gene transfer mitigates the dystrophic phenotype of mdx mouse muscles.

Utrophin is a close homolog of dystrophin, the protein whose mutations cause Duchenne muscular dystrophy (DMD). Utrophin is present at low levels in normal and dystrophic muscle, whereas dystrophin is largely absent in DMD. In such cases, the replacement of dystrophin using a utrophin gene transfer strategy could be more advantageous because utrophin would not be a neoantigen. To establish if adenovirus (AV)-mediated utrophin gene transfer is a possible option for the treatment of DMD, an AV vector expressing a shortened version of utrophin (AdCMV-Utr) was constructed. The effect of utrophin overexpression was investigated following intramuscular injection of this AV into mdx mice, the mouse model of DMD. When the tibialis anterior (TA) muscles of 3- to 5-day-old animals were injected with 5 microl of AdCMV-Utr (7.0 x 10(11) virus/ml), an average of 32% of fibers were transduced and the transduction level remained stable for at least 60 days. The presence of utrophin restored the normal histochemical pattern of the dystrophin-associated protein complex at the cell surface and resulted in a reduction in the number of centrally nucleated fibers. The transduced fibers were largely impermeable to the tracer dye Evans blue, suggesting that utrophin protects the surface membrane from breakage. In vitro measurements of the force decline in response to high-stress eccentric contractions demonstrated that the muscles overexpressing utrophin were more resistant to mechanical stress-induced injury. Taken together, these data indicate that AV-mediated utrophin gene transfer can correct various aspects of the dystrophic phenotype. However, a progressive reduction in the number of transduced fibers was observed when the TA muscles of 30- to 45-day-old mice were injected with 25 microl of AdCMV-Utr. This reduction coincides with a humoral response to the AV and transgene, which consists of a hybrid mouse-human cDNA.

Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15-17-fold increase of DRP over normal in DMD/BMD and 4-10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.