ABSTRACT: Ullrich’s disease is a congenital muscular dystrophy charac-terized clinically by generalized muscle weakness, multiple contractures ofthe proximal joints, and hyperextensibility of the distal joints. Recent studieshave demonstrated that collagen VI is deficient in the muscles of patientswith Ullrich’s disease, and some cases result from recessive mutations ofthe collagen VI�2 gene (COL6A2). Fibronectin is one of the main compo-nents of the extracellular matrix (ECM) and associates with a variety of othermatrix molecules including collagen. The behavior of fibronectin on cells ismediated by fibronectin receptors, members of the integrin family. Westudied the expression of fibronectin receptors and fibronectin in patientswith Ullrich’s disease, and found a marked reduction of fibronectin receptorsin the ECM of skin and cultured fibroblasts of these patients. These resultssuggest that collagen VI deficiency may lead to the reduction of fibronectinreceptors and that an abnormality of cell adhesion may be involved in thepathogenesis of Ullrich’s disease.

© 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 696–701, 2002

FIBRONECTIN RECEPTOR REDUCTION IN SKIN ANDFIBROBLASTS OF PATIENTS WITH ULLRICH’S DISEASEJING HU, MD,1 ITSURO HIGUCHI, MD,1 TADAFUMI SHIRAISHI, MD,1 MASAHITO SUEHARA, MD,2

TAKAHITO NIIYAMA, MD,1 TAKASHI HORIKIRI, MD,1 YUICHI UCHIDA, MD,1

AKIKO SAITO, MD,1 and MITSUHIRO OSAME, MD1

1 Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka,Kagoshima 890-8520, Japan2 Department of Neurology, National Okinawa Hospital, Okinawa, Japan

Accepted 5 July 2002

Fibronectin is a multifunctional extracellular matrix(ECM) and plasma protein that plays a central rolein cell adhesion.24 It is a dimeric glycoprotein thatundergoes polymerization into insoluble ECM fibrilsin various connective tissues.4,26 Recently, the focusof fibronectin research has shifted to its cell surfacereceptors and their relationship to other related ad-hesion receptors, collectively called integrins. Fi-bronectin binds to the cell surface via an integrinreceptor, usually �5�1 integrin,3,10 and triggers theactivation of signaling pathways leading to cy-toskeletal reorganization, cell cycle progression,27

and differentiation.8 Many other integrins, includ-ing �3�1,28 �4�1,9 and �IIb�3,22 interact with fi-bronectin as well as other components of the ECM.Among them, �5�1 integrin exhibits the highest

affinity to fibronectin, and it appears to be theonly integrin receptor that interacts exclusivelywith fibronectin.

Collagen VI is also a major ECM protein and con-sists of three genetically distinct � chains. The hetero-trimer (�1,�2,�3) is made up of two large globulardomains connected by a triple helical stalk.1,5 CollagenVI has been shown to bind to cells,6 and to interact withseveral ECM components including fibrillar collagens Iand II, collagen IV, fibronectin, biglycan, anddecorin.19,29,31 It forms a microfilamentous network,which is particularly abundant close to the cells andwhich has been suggested to anchor the basementmembrane to the underlying connective tissue.18

Heterozygous mutations of the genes that codefor collagen VI subunits have been reported in au-tosomal dominant Bethlem myopathy.17,21 Ullrich’sdisease is a congenital disorder described by Ullrichin 1930. The major clinical findings include gener-alized muscle weakness and wasting, striking contrac-tures of the proximal joints, hyperflexibility of thedistal joints from an early infantile stage, and a pro-gressive course. A deficiency of collagen VI proteinin patients with Ullrich’s disease and recessive mu-tations in the collagen VI�2 gene (COL6A2) leadingto Ullrich’s disease have been reported.2,14,15

Abbreviations: COL6A2, collagen VI�2 gene; DMEM, Dulbecco’s modifiedEagle’s medium; ECM, extracellular matrix; FBS, fetal bovine serumKey words: collagen VI, congenital muscular dystrophy, fibronectin, fi-bronectin receptor, Ullrich’s diseaseCorrespondence to: I. Higuchi; e-mail: ihiguchi@m2.kufm.kagoshima-u.ac.jp

© 2002 Wiley Periodicals, Inc.Published online 30 September 2002 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.10250

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In the present study, we investigated the expres-sion of fibronectin and its receptors in skin andcultured fibroblasts of patients with Ullrich’s disease.The purposes of this study were to determine thedistributions and interactions of fibronectin, fi-bronectin receptors, and collagen VI in skin andcultured fibroblasts, and to elucidate the pathogen-esis of Ullrich’s disease.

MATERIALS AND METHODS

We examined two patients with Ullrich’s disease, inboth of whom a deficiency of collagen VI has previ-ously been reported14 and in one of whom (patient1) a recessive mutation of COL6A2 has already beenreported.15 The gene mutation in the other patient(patient 2) has not yet been determined. Frozenbiopsied skin specimens from the patients with Ull-rich’s disease and also from six patients with otherneuromuscular diseases (three patients with Duch-

enne muscular dystrophy, two patients with Beckermuscular dystrophy, and one patient with sarcogly-canopathy) were cut into 8 �m sections. An immu-nohistochemical study was performed on compo-nents of the ECM. The monoclonal antibodies usedcomprised a 1:100 dilution of fibronectin-receptor(VLA5) (Cosmo-Bio, Tokyo, Japan), a 1:1,000 dilu-tion of collagen VI (Fuji Chemical, Takaoka, Japan),and a 1:50 dilution of fibronectin (Chemicon, Te-mecula, California); all of the immunohistochemicalprocedures were performed as reported previously.13

Fibroblasts were obtained from biopsied skinfrom the patients with Ullrich’s disease and fromfour patients with other neuromuscular diseases in-cluding two patients with Duchenne muscular dys-trophy, one patient with Becker muscular dystrophy,and one patient with myasthenia gravis. Cell cultureswere performed as follows. First, fibroblasts wereplated on culture dishes (diameter, 100 mm), and

FIGURE 1. Immunohistochemical analysis of fibronectin-receptors (A–C), collagen VI (D–F), and fibronectin (G–I) in biopsied skinspecimens from a patient with Duchenne muscular dystrophy (A,D,G), and patient 1 (B,E,H) and patient 2 with Ullrich’s disease (C,F,I).Expression of fibronectin receptors and collagen VI is reduced or deficient in the dermis of the patients with Ulrich’s disease. Fibronectinis slightly more increased in the dermis in patient 1 (H) than patient 2 with Ullrich’s disease (I) and the control patient (G). Originalmagnification, �100.

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cultured in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal bovine serum (FBS);the medium was changed on the next day and thenevery 2 days until 80% of the fibroblasts reached theconfluent phase, which was after 7–10 days. Second,the fibroblasts were retrieved with trypsin and seededinto chamber slides with 10% FBS DMEM; the medium(2% horse serum containing DMEM) was changed onthe next day and then every 2 days, and the fibroblastsunderwent differentiation within 5–7 days. The fibro-blasts were cultured in an incubator (37°C, 5% CO2).Finally, the cells were fixed with 50% acetone and 50%methanol (�20°C, 10 min), and then washed with PBS(4°C, 5 min) for immunohistochemistry.

RESULTS

The results of immunohistochemical analysis of skinare presented in Figure 1. In the skin of the sixcontrol patients with Duchenne muscular dystrophy,Becker muscular dystrophy, or sarcoglycanopathy,strong expression of fibronectin receptors was ob-

served in the epidermis and dermis. In patients withUllrich’s disease, however, the expression of fi-bronectin receptors was observed only in the epider-mis, and was markedly reduced in the ECM of thedermis. In the control patients, collagen VI was notobserved in the epidermis, but was strongly ex-pressed in the ECM of the dermis. In patients withUllrich’s disease, collagen VI was deficient in the ECMof the dermis. Thus, both fibronectin receptors andcollagen VI were reduced in the ECM of the dermis ofthe two patients with Ullrich’s disease. Although fi-bronectin expression was not markedly different be-tween Ullrich’s disease patients and the other patients,patient 1 with Ullrich’s disease showed slightly in-creased expression of fibronectin in the dermis com-pared to the control patients and patient 2 with Ull-rich’s disease.

The results of immunohistochemical analysis ofcultured fibroblasts are presented in Figure 2. Incultured fibroblasts of the patients with Ullrich’sdisease, the expression of fibronectin receptors and

FIGURE 2. Immunohistochemical analysis of fibronectin-receptors (A–C), collagen VI (D–F), and fibronectin (G–I) in cultured fibroblastsfrom a patient with myasthenia gravis (A,D,G), and patient 1 (B,E,H) and patient 2 with Ullrich’s disease (C,F,I). Both fibronectin receptorsand collagen VI are markedly reduced in the cultured fibroblasts of the patients with Ullrich’s disease. The reduction in patient 1 (B,E) ismore marked than that in patient 2 (C,F). Original magnification, �400.

698 Fibronectin Receptor Reduction MUSCLE & NERVE November 2002

collagen VI was markedly decreased. However, incontrast to the skin, the extent of reduction in fi-bronectin receptors and collagen VI was more prom-inent in patient 1 than patient 2 with Ullrich’s dis-ease. However, fibronectin was strongly expressed inthe fibroblasts of the two patients with Ullrich’s dis-ease and the control patients.

During the fibroblast culture and staining pro-cess, we noted that fibroblasts from patients withUllrich’s disease became detached from non-coatedchamber slide glasses more easily than those of con-trol patients. This detachment was prevented by us-ing gelatin-coated chamber slide glasses (Fig. 3).

DISCUSSION

On immunohistochemical examination, we demon-strated that a similar amount or more fibronectin is

secreted and deposited in the skin and culturedfibroblasts in Ullrich’s disease patients than in con-trol patients with other neuromuscular diseases. Inaccordance with our results, it has been reportedthat the fibronectin level and molecular weight infibroblasts from collagen VI�1 null mice were similarto those in controls.25 However, fibronectin recep-tors were markedly reduced in the skin and culturedfibroblasts of patients with Ullrich’s disease and col-lagen VI deficiency than in control diseases. Theseresults suggest that collagen VI deficiency may leadto the reduction of fibronectin receptors in the skinand cultured fibroblasts.

Collagen VI mediates the three-dimensional or-ganization of fibronectin in the ECM of culturedfibroblasts, and it has been reported that collagen VIdeficiency affects the organization of fibronectin in

FIGURE 3. Hematoxylin and eosin staining in cultured fibroblasts from a patient with myasthenia gravis (A,B), and patient 1 (C,D) andpatient 2 with Ullrich’s disease (E,F). Ullrich’s disease fibroblasts become detached from non-coated chamber slide glasses (D,F) moreeasily than those of a control patient (B). This detachment is prevented by using gelatin-coated chamber slide glasses (C,E).Gelatin-coated chamber slide glasses (A,C,E); non-coated chamber slide glasses (B,D,F). Original magnification, �150.

Fibronectin Receptor Reduction MUSCLE & NERVE November 2002 699

the ECM of cultured fibroblasts from collagen VI�1null mutant mice and from a patient with Bethlemmyopathy.25 It has also been reported that fibronec-tin interacts with collagen VI via its receptor.16 Thecell attachment site has been identified in culturedcells and tissues by affinity chromatography of fi-bronectin cell attachment fragments16,23 or in a lessdirect way by means of antibodies that inhibit theattachment of cells to fibronectin.11 Cell adhesion toECM proteins is essential for organogenesis, devel-opment, differentiation, wound healing, and tissuehomeostasis and remodeling.7,20 The reduction offibronectin receptors in Ullrich’s disease suggeststhat abnormal adhesion of fibroblasts to the ECMmay be involved in the pathogenesis of Ullrich’sdisease. The abnormality of cell adhesion in Ull-rich’s disease was also demonstrated by the easy de-tachment of fibroblasts from the chamber slideswithout a gelatin coating.

The reduction of collagen VI and fibronectinreceptor was similar in the skin but different in thecultured fibroblasts between the two patients withUllrich’s disease. Fibronectin receptors and collagenVI were absent in cultured fibroblasts of patient 1with recessive mutations of COL6A2. However, theywere partly expressed in the cultured fibroblasts ofpatient 2. This indicates that fibronectin receptorsand collagen VI protein may remain in immaturecells in Ullrich’s disease, and that the two patientsmay have different genetic backgrounds. It willtherefore be important to determine the gene mu-tation of collagen VI in patient 2.

Collagen VI shows a widespread distribution invirtually all connective tissues, including skeletalmuscle,12 joint capsules, ligaments, tendons, andskin.30 Although the skin in Ullrich’s disease withcollagen VI deficiency shows hyperextensibility, theskin symptoms are milder than those of skeletal mus-cle. The expression of fibronectin receptors in skel-etal muscle fibers was faint even in the control skel-etal muscle fibers. Therefore, it is difficult to detecta reduction of it in skeletal muscle of patients withUllrich’s disease (data not shown). The expressionof fibronectin receptors in the vessel walls within theskeletal muscle did not differ between the controlpatients and those with Ullrich’s disease. As it ispossible that other adhesion molecules may be mod-ified by the deficiency of collagen VI, further study isnecessary to elucidate the different alterations ofECM components in muscle and other tissues. Asmental and cardiac functions are normal in mostpatients with Ullrich’s disease, it is suggested that notonly the distribution of collagen VI but also its rolemay differ among different tissues.

In conclusion, our findings suggest that collagenVI deficiency may lead to the reduction of fibronec-tin receptors and abnormal adhesion of fibroblastsin Ullrich’s disease, and that these findings may beinvolved in the pathogenesis of this disease.

This study was supported in part by a Research Grant (11B-1) forNervous and Mental Disorders from the Ministry of Health, La-bour, and Welfare. We thank Noriko Hirata for her technicalassistance.

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