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Keywords:

  • collagen VI;
  • fibronectin receptor;
  • NG2 proteoglycan;
  • tenascin C;
  • Ullrich's disease

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients with Ullrich's disease have generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. Recently we found a marked reduction of fibronectin receptors in the skin and cultured fibroblasts of two patients with Ullrich's disease with collagen VI deficiency, and speculated that an abnormality of cell adhesion may be involved in the pathogenesis of the disease. In this study, we investigated the expression of proteoglycans and adhesion molecules in Ullrich's disease and other muscle diseases. We found a reduction of NG2 proteoglycan in the membrane of skeletal muscle but not in the skin in Ullrich's disease. By contrast, we found the upregulation of tenascin C in the extracellular matrix of skeletal muscle in Ullrich's disease. Our findings suggest that abnormal expression of proteoglycans and adhesion molecules may be involved in the pathogenesis of the dystrophic muscle changes in Ullrich's disease. Muscle Nerve, 2006

Ullrich's disease or Ullrich congenital muscular dystrophy (UCMD) is a unique congenital disorder that was described as congenital hypotonic-sclerotic muscular dystrophy by Ullrich.33 The major clinical findings include generalized muscle weakness and wasting, striking contractures of the proximal joints and hyperflexibility of the distal joints from an early infantile stage, and a progressive course. Recently, we found a deficiency of collagen VI protein in skeletal muscle from two patients with Ullrich's disease.16 Subsequently, we and other groups detected recessive mutations in the collagen VI genes in Ullrich's disease.5, 7, 15 A heterozygous in-frame deletion in the collagen VI alpha 1 gene also causes Ullrich's disease.1, 27

Collagen VI has been shown to bind to cells, and to interact with several extracellular matrix (ECM) components including collagen types I,35 II,2 IV,20 and XIV,3 fibronectin,32 fibromodulin,12 hyaluronic acid,22 perlecan,32 decorin,32 biglycan,37 and NG2 proteoglycan.26, 30 It forms a microfilamentous network, which is particularly abundant close to the cells and which has been suggested to anchor the basement membrane to the underlying connective tissue.19

Although it has been demonstrated that a heritable collagen disorder can cause congenital muscular dystrophy, the precise mechanism underlying the dystrophic muscle changes and characteristic clinical manifestations is unknown. In this study, we investigated immunohistochemically the expression of proteoglycans and adhesion molecules in Ullrich's disease to determine whether abnormal expression of proteoglycans and adhesion molecules is involved in the pathogenesis of Ullrich's disease. Furthermore, we compared the immunohistochemical staining of these proteins and remaining collagen VI protein in different tissues to elucidate the cause of the different severities of clinical manifestations in different tissues in Ullrich's disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients.

We have previously reported the clinical details of two male patients (patient 1: 20 years old; patient 2: 21 years old) with sporadic Ullrich's disease, who have a frameshift mutation in the triple-helical domain of collagen VI alpha 2 gene.15 As controls, muscle specimens were obtained from patients with other neuromuscular disorders, including Fukuyama-type congenital muscular dystrophy (two patients), Duchenne muscular dystrophy (three patients), Becker muscular dystrophy (three patients), and polymyositis (three patients), and control patients with minimal pathological changes (three patients with fibromyalgia syndrome). The diagnoses were established with clinical, neurophysiological, and pathological criteria. The biopsied muscle specimens were immediately frozen in isopentane cooled with liquid nitrogen. Serial frozen sections (8-μm thick) were stained with hematoxylin and eosin and a battery of other histochemical stains.

Immunohistochemistry.

Frozen biopsied biceps brachii muscle and skin specimens from the patients with Ullrich's disease and other neuromuscular disorders were cut into 8-μm sections, which were picked up on aminosilane-coated slides. An immunohistochemical study was performed on the components of the ECM in the biopsied skeletal muscle and skin specimens. The polyclonal antibodies used comprised a 1:100 dilution of NG2 proteoglycan (Chemicon, Temecula, California; number AB 5320), and a 1:200 dilution of tenascin C (Chemicon). The monoclonal antibodies used comprised a 1:100 dilution of CD44 (Novocastra, Newcastle, United Kingdom), a 1:200 dilution of heparan sulfate proteoglycan (Boehringer Mannheim, Mannheim, Germany), a 1:1000 dilution of the triple-helical domain of collagen VI (Daiichi Fine Chemical, Takaoka, Japan), a 1:100 dilution of nontriple-helical domain of collagen VI (Chemicon; number MAB 1944), a 1:50 dilution of dystrophin C terminus (Novocastra), and a 1:5000 dilution of laminin B1(Chemicon). Biotinylated anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG was used as a secondary antibody, and the ABC method was used for signal detection (ABC kit; Vector, Burlingame, California). All immunohistochemical procedures were performed as reported previously.14

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

On immunohistochemical examination with the antibody to NG2 proteoglycan, the expression in the membrane of skeletal muscle was observed to increase in other neuromuscular disorders including Fukuyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, and polymyositis. In particular, dystrophin-negative fibers and regenerating fibers showed marked upregulation of NG2 proteoglycan on the sarcolemma. However, a reduction of NG2 proteoglycan was observed on the sarcolemma in small-calibered regenerating fibers (Fig. 1) but not in the ECM of skin of the two patients with Ullrich's disease. In general, NG2 proteoglycan expression decreased with advancing age.

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Figure 1. Immunohistochemical staining for NG2 proteoglycan (AD), laminin B1 (E), and C terminus of dystrophin (F) of biopsied muscle specimens from a patient with Ullrich's disease (A, E, F), a patient with Duchenne muscular dystrophy (B), an age-matched patient with Becker muscular dystrophy (C), and an age-matched control patient (D). Reduction of NG2 proteoglycan can be seen in the membrane of skeletal muscle of the patient with Ullrich's disease even in regenerating fibers (A). By contrast, increased expression of NG2 proteoglycan can be seen in other diseases (B, C). The expression of NG2 proteoglycan in vessels (AD) does not differ among the patients. Laminin B1 (E) and dystrophin (F) are normally expressed in Ullrich's disease. ×150 (original magnification).

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The tenascin C expression was upregulated in the ECM of the skeletal muscle specimens from the two patients with Ullrich's disease (Fig. 2). Unlike the other neuromuscular disorders, where tenascin expression was upregulated in the ECM with macrophage invasion, upregulation was observed in endomysium without macrophage invasion in Ullrich's disease. The expression of CD44 was increased in the membrane of skeletal muscle in various neuromuscular disorders, especially in regenerating fibers. In contrast to these other neuromuscular disorders, the expression of CD44 was not increased in the small-calibered regenerating muscle fibers in the two patients with Ullrich's disease (Fig. 3). The expression of heparan sulfate proteoglycan was upregulated in the muscle membrane in all neuromuscular disorders including Ullrich's disease.

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Figure 2. Immunohistochemical staining for tenascin C of biopsied muscle specimens from a patient with Ullrich's disease (A), a patient with Becker muscular dystrophy (B), a patient with polymyositis (C), and a control patient (D). Tenascin expression was upregulated in the extracellular matrix in polymyositis with macrophage invasion (C). However, the upregulation of tenascin in Ullrich's disease was observed in the endomysium without macrophage invasion (A). Tenascin expression was not upregulated in the extracellular matrix in Becker muscular dystrophy without macrophage invasion (B). ×150 (original magnification).

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Figure 3. Immunohistochemical staining for CD44 of biopsied muscle specimens from a patient with Ullrich's disease (A), a patient with Becker muscular dystrophy (B), a patient with Duchenne muscular dystrophy (C), and a control patient (D). In contrast to in the other muscular dystrophies (B, C), the expression of CD44 was not increased in the small-calibered regenerating muscle fibers in Ullrich's disease (A). ×150 (original magnification).

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On immunohistochemical examination with the two kinds of antibodies for collagen VI in the skin in Ullrich's disease, the expression of collagen VI was absent in the dermis. However, the remaining collagen VI protein was observed in arrector pili muscles and myoepithelial cells of sweat glands. Furthermore, immunoreactivity for the antibody against nontriple-helical domain of collagen VI was upregulated in the epidermis in Ullrich's disease in contrast to the deficiency of that in the dermis (Fig. 4).

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Figure 4. Immunohistochemical staining of biopsied skin specimens from a patient with Ullrich's disease (A, C, E) and a patient with Duchenne muscular dystrophy (B, D, F). Slight upregulation of NG2 proteoglycan in the skin from a patient with Ullrich's disease (A) can be seen. Normal expression of collagen VI in the dermis can be seen with the two kinds of antibodies in Duchenne muscular dystrophy. On immunohistochemical staining with a monoclonal antibody for the triple-helical domain of collagen VI, the expression of collagen VI is absent in Ullrich's disease except for arrector pili muscles (arrowhead) and myoepithelial cells of sweat glands (C). On immunohistochemical staining with a monoclonal antibody for nontriple-helical domain of collagen VI, the upregulation of collagen VI can be seen in the epidermis in Ullrich's disease (arrows) in contrast to the deficiency of that in the dermis (E). (A, B) Immunohistochemical staining for NG2 proteoglycan, ×150 (original magnification). (C, D) Immunohistochemical staining with a monoclonal antibody for triple helical domain of collagen VI, ×150 (original magnification). (E, F) Immunohistochemical staining with a monoclonal antibody for nontriple-helical domain of collagen VI, ×150 (original magnification).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In our previous study, we found a marked reduction of fibronectin receptors in the skin and cultured fibroblasts of two patients with Ullrich's disease with collagen VI deficiency. We speculated that collagen VI deficiency may lead to a reduction of fibronectin receptors and that an abnormality of cell adhesion may be involved in the pathogenesis of Ullrich's disease.17 Recently, we evaluated the role of nonsense-mediated mRNA decay (NMD) in Ullrich's disease and found that the pharmacological block of NMD caused upregulation of the mutant collagen VI, resulting in upregulation of the fibronectin receptor and partially functional ECM formation.34 The expression of fibronectin receptors in skeletal muscle fibers was faint even in the control skeletal muscle fibers. Therefore, it was difficult to detect a reduction of it in the skeletal muscle of patients with Ullrich's disease.

In this study, we investigated immunohistochemically the expression of proteoglycans and adhesion molecules other than fibronectin and fibronectin receptor in Ullrich's disease. We found a reduction of NG2 proteoglycan in the membrane of skeletal muscle but not in the vessels or ECM of the skin of these patients. Petrini et al. have reported that NG2 proteoglycan expression decreases with advancing age in muscle biopsy specimens from normal subjects, and that dystrophin-negative fibers in Duchenne and Becker muscular dystrophy, and regenerating muscle fibers in sarcoglycanopathy and calpainopathy, show strong NG2 proteoglycan expression.28 They also demonstrated that NG2 proteoglycan localization on the sarcolemma is markedly decreased in muscle fibers in merosin-deficient congenital muscular dystrophy (MDC1A), whereas regenerating fibers and blood vessels show normal expression. In the present study, the reduction of NG2 proteoglycan in the membrane of skeletal muscle in Ullrich's disease was observed even in regenerating fibers. Further Western blot and Northern blot studies are necessary to determine whether the reduced expression of NG2 proteoglycan is due to a transcriptional or translational block.

NG2 proteoglycan is a 500-kDa integral membrane chondroitin sulfate proteoglycan that is widely expressed on various cell types. Although the exact function of NG2 proteoglycan is not clear, several cell lines reportedly coexpress NG2 proteoglycan and collagen VI in an identical pattern on the cell surface. Furthermore, retention of collagen VI on cells could be inhibited with specific antisera against NG2 proteoglycan. These studies strongly suggested the close interaction of the two molecules.26, 30 The present study on Ullrich's disease also suggests the close relation of collagen VI and NG2 proteoglycan on the muscle cell membrane.

NG2 proteoglycan has the ability to enhance cellular responses to platelet-derived growth factor (PDGF)-AA,10 and the PDGF alpha-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from NG2 knockout mice.9 Furthermore, NG2 proteoglycan is reported to mediate beta1 integrin-independent cell adhesion and to trigger signaling events that lead to rearrangement of the actin cytoskeleton.31 The reduction of NG2 proteoglycan in the skeletal muscle in Ullrich's disease might influence the development or regeneration of muscle fibers through a defect of signal transduction. However, NG2 proteoglycan has been reported to be an axon-inhibitory proteoglycan in the central nervous system8 and peripheral nerves.21 Further studies on the expression of NG2 proteoglycan in various tissues are necessary to elucidate the pathological role of the reduction of NG2 proteoglycan in Ullrich's disease. We have previously examined the skeletal muscle of Ullrich's disease by electron microscopy and found unevenness, extension, and folding of the muscle plasma membrane.25 The reduction of NG2 proteoglycan in the muscle membrane in Ullrich's disease may relate to these electron microscopic abnormalities.

The second characteristic finding in the present study is the upregulation of tenascin C in the ECM of skeletal muscle in Ullrich's disease. Tenascin C modulates the adhesion of cells to fibronectin and can be classified as an adhesion-modulating ECM protein.6, 29 Tenascin C is expressed during development and is reduced in most mature organs, but reappears under pathological conditions such as inflammation, infection, and tumorigenesis.18 In skeletal muscle, confluent tenascin expression in the extracellular space in dermatomyositis and expression confined to the surroundings of necrotic fibers in polymyositis have been reported.23 Further, the endomysial tenascin C reactivity in Duchenne/Becker muscular dystrophy and myositis is invariably correlated with the presence of macrophages.11 However, in the present study, tenascin C expression in Ullrich's disease did not correlate with the presence of macrophages. A report on binding studies has revealed several possible ligands for NG2 proteoglycan other than collagen VI, including type II collagen, type V collagen, laminin, and tenascin.4 It is possible that the upregulation of tenascin C in the ECM compensates for the collagen VI deficiency or NG2 proteoglycan reduction in Ullrich's disease.

However, the upregulation of tenascin and the abnormal expression of CD44 might be related to reduced cell adhesion and abnormal regeneration in Ullrich's disease. Thus, collagen VI deficiency causes abnormalities of proteoglycans and ECM, which may be linked to the dystrophic muscle changes in Ullrich's disease.

Interestingly, the expression of NG2 proteoglycan and tenascin differs among different tissues in Ullrich's disease. The reduction of NG2 proteoglycan and the upregulation of tenascin were not observed in the skin. Such differences in the expression of proteoglycans between the different tissues might be one of the causes of the different severities of clinical manifestations in different tissues, from which collagen VI is absent. The present findings suggest that abnormal expression of proteoglycans and adhesion molecules are involved in the abnormality of ongoing regeneration or complete maturation of muscle fibers13, 24 in Ullrich's disease.

Concerning the collagen VI upregulation in the epidermis in Ullrich's disease, as found with the antibody against the nontriple-helical domain of collagen VI, we could not undertake immunoblotting analysis because of the small amount of biopsied skin sample. However, we think this is a specific finding because the finding was not observed in the skin of other diseases and controls. We speculate that this antibody might react to certain incomplete collagen VI molecules. The variant collagen VI alpha 3 chain has been detected in the epidermis in contrast to the usual collagen VI expression in the dermis in photoaged skin.36 Thus, the variable severity in different tissues in Ullrich's disease may be related to a number of factors including differences in remaining collagen VI protein, different expression of collagen VI-associated molecules such as NG2 proteoglycan, and compensatory expression of other collagen molecules or unknown proteins.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

This study was supported in part by a research grant (11B-1) for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare. We thank Noriko Hirata for her technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES