Activation of MAPK in hearts of EMD null mice: similarities between mouse models of X-linked and autosomal dominant Emery Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is an inherited disorder characterized by slowly progressive skeletal muscle weakness in a humero-peroneal distribution, early contractures and prominent cardiomyopathy with conduction block. Mutations in EMD, encoding emerin, and LMNA, encoding A-type lamins, respectively, cause X-linked and autosomal dominant EDMD. Emerin and A-type lamins are proteins of the inner membrane of the nuclear envelope. Whereas the genetic cause of EDMD has been described and the proteins well characterized, little is known on how abnormalities in nuclear envelope proteins cause striated muscle disease. In this study, we analyzed genome-wide expression profiles in hearts from Emd knockout mice, a model of X-linked EDMD, using Affymetrix GeneChips. This analysis showed a molecular signature similar to that we previously described in hearts from Lmna H222P knock-in mice, a model of autosomal dominant EDMD. There was a common activation of the ERK1/2 branch of the mitogen-activated protein kinase (MAPK) pathway in both murine models, as well as activation of downstream targets implicated in the pathogenesis of cardiomyopathy. Activation of MAPK signaling appears to be a cornerstone in the development of heart disease in both X-linked and autosomal dominant EDMD.     

The role of branched fibres in the pathogenesis of Duchenne muscular dystrophy

Branched fibres are a well-documented phenomenon of regenerating skeletal muscle. They are found in the muscles of boys with Duchenne muscular dystrophy (DMD), a severe condition of progressive muscle wasting caused by an absence of the sarcolemmal protein dystrophin, and in the muscles of the mdx mouse, an animal model of DMD. However, only a handful of studies have investigated how the physiological properties of these morphologically deformed fibres differ from those of normal fibres. These studies have found an association between the extent of fibre branching in mdx muscles and the susceptibility of these muscles to damage from eccentric contractions. They have also found that branched mdx muscle fibres cannot sustain maximal contractions in buffered Ca(2+) solutions, that branch points are sites of increased mechanical stress and that myofibrillar structure is greatly disturbed at branch points. These findings have important implications for understanding the function of dystrophin. It is commonly thought that the role of dystrophin is mechanical stabilization of the sarcolemma, as numerous studies have shown that eccentric contractions damage mdx muscle more than normal muscle. However, the finding that branched mdx fibres are mechanically weakened raises the question, is it the lack of dystrophin or is it the fibre branching that leads to the vulnerability of mdx muscle to contractile damage? The importance of this question to our understanding of the function of dystrophin warrants further research into the physiological properties of branched fibres and how they differ from morphologically normal fibres.     

Novel LMNA mutations in patients with Emery‐Dreifuss muscular dystrophy and functional characterization of four LMNA mutations

Mutations in LMNA cause a variety of diseases affecting striated muscle including autosomal Emery‐Dreifuss muscular dystrophy (EDMD), LMNA‐associated congenital muscular dystrophy (L‐CMD), and limb‐girdle muscular dystrophy type 1B (LGMD1B). Here, we describe novel and recurrent LMNA mutations identified in 50 patients from the United States and Canada, which is the first report of the distribution of LMNA mutations from a large cohort outside Europe. This augments the number of LMNA mutations known to cause EDMD by 16.5%, equating to an increase of 5.9% in the total known LMNA mutations. Eight patients presented with either p.R249W/Q or p.E358K mutations and an early onset EDMD phenotype: two mutations recently associated with L‐CMD. Importantly, 15 mutations are novel and include eight missense mutations (p.R189P, p.F206L, p.S268P, p.S295P, p.E361K, p.G449D, p.L454P, and p.W467R), three splice site mutations (c.IVS4 + 1G>A, c.IVS6 ? 2A>G, and c.IVS8 + 1G>A), one duplication/in frame insertion (p.R190dup), one deletion (p.Q355del), and two silent mutations (p.R119R and p.K270K). Analysis of 4 of our lamin A mutations showed that some caused nuclear deformations and lamin B redistribution in a mutation specific manner. Together, this study significantly augments the number of EDMD patients on the database and describes 15 novel mutations that underlie EDMD, which will contribute to establishing genotype–phenotype correlations. Hum Mutat 31:–16, 2011. © 2011 Wiley‐Liss, Inc.     

The functional polymorphisms of VDR,GC and CYP2R1 are involved in the pathogenesis of autoimmune thyroid diseases

Vitamin D is a multi-functional immune regulator, and a low serum concentration of vitamin D promotes autoimmune inflammation. In this study, we evaluate the association between the prognosis of autoimmune thyroid disease (AITD) and the functional polymorphisms of genes that regulate vitamin D metabolism. For 139 Graves’ disease (GD) patients, 116 Hashimoto''s disease (HD) patients and 76 control subjects, we genotyped the following polymorphisms using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP): vitamin D receptor (VDR): rs731236, rs7975232, rs2228570 and rs1544410; group-specific component (GC): rs7041 and rs4588; and CYP2R1: rs10741657. The frequency of the TT genotype for the rs731236 polymorphism was higher in GD patients than in HD patients (P = 0·0147). The frequency of the C allele for the rs7975232 polymorphism was higher in GD patients than in control subjects (P = 0·0349). The proportion of GD patients whose anti-thyrotrophin receptor antibody (TRAb) level was >51% was higher in those with the CC genotype than in those with the CA+AA genotypes (P = 0·0065). The frequency of the CC genotype for the rs2228570 polymorphism was higher in HD patients than in control subjects (P = 0·0174) and GD patients (P = 0·0149). The frequency of the Gc1Gc1 genotype for the GC polymorphism and the AG genotype for the CYP2R1 polymorphism were lower in intractable GD than in GD in remission (P = 0·0093 and 0·0268, respectively). In conclusion, genetic differences in the VDR gene may be involved in the development of AITD and the activity of GD, whereas the genetic differences in the GC and CYP2R1 genes may be involved with the intractability of GD.     

计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病

背景：Duchenne型肌营养不良和Becker型进行性肌营养不良都是dystrophin基因突变所致,但后者临床表型较轻。“阅读框规则”可解释大部分基因型与临床型关系,但累及疏水区段的整码突变也可导致Duchenne型肌营养不良。因此很有必要了解疏水区域在dystrophin中的功能,且这些疏水区段的三维结构及功能在发病机制中所起的具体作用仍未阐明。 目的：通过Kyte&Doolittle平均疏水轮廓分析研究dystrophin的疏水区段。利用swiss-model三维重构dystrophin的疏水区段阐述其在发病机制中所起的作用。 方法：参考莱顿开放数据库(http://www.dmd.nl/)及收集中山大学附属第一医院2002年至2013年确诊Duchenne型进行性肌营养不良或Becker型进行性肌营养不良的缺失型整码突变患者资料共1 038例,分析其临床型与基因型关系。使用bioedit软件计算dystrophin的平均疏水轮廓及利用swiss-model三维重构疏水区段,结合临床型和基因型关系确定dystrophin重要功能区。 结果与结论：dystrophin存在4个疏水区段,分别为肌动蛋白结合区内的Calponin同源区2、中央棒区内的重复区16、第三铰链区和EF手型区。第1,2,4疏水区段是dystrophin糖蛋白复合物中dystrophin与其他糖蛋白的结合区域,其破坏严重影响dystrophin糖蛋白复合物功能,临床症状重。中央棒区在第三铰链区附近断裂后,HⅢ的无规则卷结构不容易与断端重复区的螺旋结构恢复连接。但第三铰链区同时缺失,其两端的重复区较容易重新连接,所以第3疏水区破坏后其临床症状反而较轻。提示dystrophin的疏水区段是其重要功能区,多是dystrophin糖蛋白复合物中dystrophin与相关蛋白的结合部位,在Duchenne型肌营养不良的发病机制中起重要作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Localization of interferons and interleukin 2 in polymyositis and muscular dystrophy

Muscle biopsies from nine patients with polymyositis, six with muscular dystrophy, six with other muscle diseases and three controls have been studied with a panel of 10 monoclonal antibodies (MoAb) identifying T lymphocytes, HLA-class I antigens, alpha, beta and gamma interferons and interleukin 2 (IL-2). The result confirm that the staining of the sarcolemma with anti-HLA class I antibody is weak or negative, except in areas adjacent to infiltrating leucocytes or where muscle fibre damage is evident. The very similar tissue distribution of alpha, beta and gamma interferons in the polymyositis biopsies supports the hypothesis that interferons are released by the inflammatory infiltrate and induce the class I antigen expression. In contrast, little interferon was demonstrated in the dystrophic muscle implying that class I expression in these disorders must occur by a different mechanism. Little IL-2 was demonstrated in any of the biopsies though some unexplained small dense accumulations were identified by one of the anti IL-2 MoAb.     

PTCH mutations are not mainly involved in the pathogenesis of sporadic trichoblastomas

Trichoblastomas are rare, benign tumors of the appendix in human skin. The histopathology comprises elements of basal cell carcinoma and trichoepithelioma with a variable degree of follicular differentiation. Both basal cell carcinoma and trichoepithelioma reveal alterations of PTCH, the human homolog of the Drosophila segment polarity patched gene. Furthermore, heterozygous PTCH knockout mice develop trichoblastoma-like tumors. This suggests an involvement of the PTCH gene in the pathogenesis of human trichoblastomas. However, trichoblastomas arising in nevus sebaceus did not show loss of heterozygosity at the PTCH locus (9q22.3) in a previous study. Sequencing of the PTCH gene and analysis of sporadic human trichoblastomas have not been performed yet. We therefore screened 10 sporadic trichoblastomas and 1 trichoblastoma arising within a nevus sebaceus for PTCH mutations. After microdissection of the tumors, single-strand conformational polymorphism (SSCP)/heteroduplex analysis of exons 2 to 23 of PTCH was performed, and polymerase chain reaction products with aberrant band patterns were sequenced. One trichoblastoma revealed a silent mutation at codon 562 in exon 12. Another trichoblastoma showed a somatic C > T single nucleotide substitution at codon 1,315 (exon 23), which was not present in corresponding normal epidermis. This mutation at codon 1,315 represents an already described PTCH germline polymorphism and results in a heterozygous Pro to Leu substitution in the tumor. The Pro/Leu polymorphism in germline is associated with a higher risk for breast cancer, but a potential contribution to the tumorigenesis of trichoblastoma is unknown. We detected no classical PTCH mutations in the investigated trichoblastomas. Our results indicate that PTCH mutations are not mainly involved in the pathogenesis of sporadic trichoblastomas, in contrast to basal cell carcinomas and trichoepitheliomas. The genetic basis of this rare appendageal tumor remains elusive.     

Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors

A subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na⁺/H⁺ exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced ²²Na⁺ influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca²⁺ as well as Na⁺ concentration and stretch-induced damage, suggesting that Na⁺_i-dependent Ca²⁺overload via the Na⁺/Ca²⁺ exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na⁺-dependent ion exchangers play a critical pathological role in muscular dystrophy.     

Overexpression of the cytotoxic T cell (CT) carbohydrate inhibits muscular dystrophy in the dyW mouse model of congenital muscular dystrophy 1A

A number of recent studies have demonstrated therapeutic effects of transgenes on the development of muscle pathology in the mdx mouse model for Duchenne muscular dystrophy, but none have been shown also to be effective in mouse models for laminin alpha2-deficient congenital muscular dystrophy (MDC1A). Here, we show that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) is effective in inhibiting the development of muscle pathology in the dy(W) mouse model of MDC1A, much as we had previously shown in mdx animals. Embryonic overexpression of Galgt2 in skeletal muscles using transgenic mice or postnatal overexpression using adeno-associated virus both reduced the extent of muscle pathology in dy(W)/dy(W) skeletal muscle. As with mdx mice, embryonic overexpression of the Galgt2 transgene in dy(W)/dy(W) myofibers inhibited muscle growth, whereas postnatal overexpression did not. Both embryonic and postnatal overexpression of Galgt2 in dy(W)/dy(W) muscle increased the expression of agrin, a protein that, in recombinant form, has been shown to ameliorate disease, whereas laminin alpha1, another disease modifier, was not expressed. Galgt2 over-expression also stimulated the glycosylation of a gly-colipid with the CT carbohydrate, and glycolipids accounted for most of the CT-reactive material in postnatal overexpression experiments. These experiments demonstrate that Galgt2 overexpression is effective in altering disease progression in skeletal muscles of dy(W) mice and should be considered as a therapeutic target in MDC1A.     

MicroRNA-21 and -146b are involved in the pathogenesis of murine viral myocarditis by regulating TH-17 differentiation

Viral myocarditis (VMC) is a common cardiovascular disease, and microRNAs (miRNAs) have been postulated to be involved in its pathology. Using microarrays, we observed that miRNA-21 and -146b were upregulated in a murine model of VMC. We also found that miRNA-451 was downregulated. In vivo silencing of miRNA-21 and -146b resulted in less-severe VMC. Overexpression of miRNA-451 did not ameliorate the severity of VMC. Further work revealed that inhibition of miRNA-21 and -146b decreased the expression levels of Th17 and RORγt . Overexpression of miRNA-451 had no effect on IL-17 and RORγt expression. Inhibition of miRNA-21 and -146b might ameliorate myocardium inflammation by mediating downregulation of RORγt expression, indicating that these miRNAs are involved in the pathogenesis of murine VMC.     

Peroxiredoxins are involved in the pathogenesis of multiple sclerosis and neuromyelitis optica spectrum disorder

Peroxiredoxins (PRXs) are intracellular anti-oxidative enzymes but work as inflammatory amplifiers under the extracellular condition. To date, the function of PRXs in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) is not fully understood. The aim of this study was to investigate whether PRXs play a role in the pathogenesis of MS and NMOSD. We analyzed levels of PRXs (PRX1, PRX5 and PRX6) in the cerebrospinal fluid (CSF) and serum of 16 patients with MS, 16 patients with NMOSD and 15 patients with other neurological disorders (ONDs). We identified potential correlations between significantly elevated PRXs levels and the clinical variables in patients with MS and NMOSD. Additionally, pathological analyses of PRXs (PRX1-6) in the central nervous system (CNS) were performed using the experimental autoimmune encephalomyelitis (EAE), animal model of MS. We found that serum levels of PRX5 and PRX6 in patients with MS and NMOSD were higher compared with those in patients with ONDs (P < 0·05). Furthermore, high levels of PRX5 and PRX6 were partly associated with blood–brain barrier dysfunction and disease duration in NMOSD patients. No significant elevation was found in CSF PRXs levels of MS and NMOSD. Spinal cords from EAE mice showed remarkable PRX5 staining, especially in CD45⁺ infiltrating cells. In conclusion, PRX5 and PRX6 may play a role in the pathogeneses of MS and NMOSD.     

Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy

Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium-binding proteins calsequestrin and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium-handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca²⁺-calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.     

Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.     

Aquaporin-1 and -5 are involved in the invasion and proliferation of soft tissue sarcomas

Background and aim

Recent studies of several carcinomas have reported that aquaporin possesses novel oncogenic properties. The aim of this study was to clarify the involvement of aquaporin-1 and ?5 in the proliferation, invasion and metastasis of soft tissue sarcomas.

Peroxiredoxins, thioredoxin, and Y-box-binding protein-1 are involved in the pathogenesis and progression of dialysis-associated renal cell carcinoma

Patients with end-stage renal disease are exposed to increased oxidative stress and impairment of antioxidant mechanisms. We focused on dialysis renal cell carcinoma (RCC), including epithelial hyperplasia in acquired cystic disease of the kidney (ACDK). We attempted to obtain insight into the carcinogenesis and tumor progression in terms of cellular defense mechanisms associated with oxidative stress by investigating the expression of antioxidant proteins by immunohistochemistry. We evaluated retrospectively 43 cases of dialysis RCC and, as a control group, 49 cases of sporadic RCC. Peroxiredoxin (Prx) 1, 3, 4, 5, and 6 expression in dialysis RCC was positively correlated with the duration of dialysis. In epithelial hyperplasia, in 17 cases of acquired cystic disease of the kidney, Prxs and thioredoxin were highly expressed. Moreover, in dialysis RCC, Prx 3, 4, and 5 immunoreactivity and nuclear expression of Y-box-binding protein-1 were higher than in sporadic RCC. In dialysis RCC, Prx 3, 4, and 5 immunoreactivity positively correlated with the Fuhrman nuclear grade. These data suggest that oxidative stress during dialysis enhances antioxidant activity, with an inhibiting effect on carcinogenesis. Once cancer has developed, antioxidant activity might have a stimulating effect on the progression of dialysis RCC.     

STAT1-mediated induction of Ly6c-expressing macrophages are involved in the pathogenesis of an acute colitis model

Background

The development of inflammatory bowel diseases is thought to be multifactorial, but the exact steps in pathogenesis are poorly understood. In this study, we investigated involvement of the activation of STAT1 signal pathway in the pathogenesis of an acute colitis model.

Methods

A dextran sulfate sodium-induced acute colitis model was established by using wild-type C57BL/6 mice and STAT1-deficient mice. Disease indicators such as body weight loss and clinical score, induction of cytokines, chemokines, and inflammatory cells were evaluated in the acute colitis model.

Results

Disease state was significantly improved in the acute colitis model using STAT1-deficient mice compared with wild-type mice. The induction of Ly6c-highly expressing cells in colorectal tissues was attenuated in STAT1-deficient mice. IL-6, CCL2, and CCR2 gene expressions in Ly6c-highly expressing cells accumulated in the inflamed colon tissues and were significantly higher than in Ly6c-intermediate-expressing cells, whereas TNF-α and IFN-α/β gene expression was higher in Ly6c-intermediate-expressing cells. Blockade of CCR2-mediated signaling significantly reduced the disease state in the acute colitis model.

Conclusions

Two different types of Ly6c-expressing macrophages are induced in the inflamed tissues through the IFN-α/β-STAT1-mediated CCL2/CCR2 cascade and this is associated with the pathogenesis such as onset, exacerbation, and subsequent chronicity of acute colitis.