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Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice.

ABSTRACT: The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, is usually confined to the sarcolemma at the neuromuscular junction in mature skeletal muscle fibers. Previously, we reported that adenovirus-mediated gene transfer is greatly facilitated in hemizygous transgenic mice with extrasynaptic CAR expression driven by a muscle-specific promoter. However, in the present study, when these mice were bred to homozygosity, they developed a severe myopathic phenotype and died prematurely. Large numbers of necrotic and regenerating fibers were present in the skeletal muscle of the homozygous CAR transgenics. The myopathy was further characterized by increased levels of caveolin-3 and beta-dystroglycan and decreased levels of dystrophin, dysferlin, and neuronal nitric-oxide synthase. Even the hemizygotes manifested a subtle phenotype, displaying deficits in isometric force generation and perturbed mitogen-activated protein kinase (MAPK-erk1/2) activation during contraction. There are few naturally occurring or engineered mouse lines showing as severe a skeletal myopathy as observed with ectopic expression of CAR in the homozygotes. Taken together, these findings suggest that substantial overexpression of CAR may lead to physiological dysfunction by disturbing sarcolemmal integrity (through dystrophin deficiency), impairing sarcolemmal repair (through dysferlin deficiency), and interfering with normal signaling (through alterations in caveolin-3 and neuronal nitric-oxide synthase levels).

SUBMITTER: Shaw CA

PROVIDER: S-EPMC1762479 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice.

Shaw Christian A CA Larochelle Nancy N Dudley Roy W R RW Lochmuller Hanns H Danialou Gawiyou G Petrof Basil J BJ Karpati George G Holland Paul C PC Nalbantoglu Josephine J

The American journal of pathology 20061201 6

The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, is usually confined to the sarcolemma at the neuromuscular junction in mature skeletal muscle fibers. Previously, we reported that adenovirus-mediated gene transfer is greatly facilitated in hemizygous transgenic mice with extrasynaptic CAR expression driven by a muscle-specific promoter. However, in the present study, when these mice were bred to homozygosity, they developed a severe myopath ...[more]

PMID: 17148677

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Project description:Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with ?-dystroglycan. This link extends to the extracellular matrix by ?-dystroglycan's interaction with ?-dystroglycan, which binds extracellular matrix proteins, including laminin ?2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle.To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity.The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles.The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

Project description:BACKGROUND:We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9):2088-95) and TGF-? (Cancer Res. 2006 Feb 1;66(3):1648-57) signaling negatively regulate coxsackie virus and adenovirus receptor (CAR) cell-surface expression and adenovirus uptake. In the case of TGF-?, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT), a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration. RESULTS:Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic) and MDA-MB-231 (breast) human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET) characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-?-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-?-induced mesenchymal phenotype of PANC-1 cells, TGF-? did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-? may inhibit CAR expression by regulating factor(s) that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal CAR promoter is positively regulated through conserved ETS and CRE elements. CONCLUSIONS:This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.

Dataset Information

Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice.

Publications

Simultaneous dystrophin and dysferlin deficiencies associated with high-level expression of the coxsackie and adenovirus receptor in transgenic mice.

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