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Human Molecular Genetics, 2003, Vol. 12, No. 19 2467-2479
DOI: 10.1093/hmg/ddg264
© 2003 Oxford University Press

Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased {alpha}7 integrin, utrophin and associated glycoproteins

Behzad Moghadaszadeh1, Reidar Albrechtsen1, Ling T. Guo2, Michaela Zaik3, Nobuko Kawaguchi1, Rehannah H. Borup4, Pauliina Kronqvist1, Henrik D. Schröder5, Kay E. Davies6, Thomas Voit3, Finn C. Nielsen4, Eva Engvall2 and Ulla M. Wewer1,*

1Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark, 2Burnham Institute, La Jolla, CA, USA, 3Department of Neuropediatrics, University of Essen, Essen, Germany, 4Department of Clinical Biochemistry, Rigshospitalet Copenhagen University Hospital, Copenhagen, Denmark, 5Department of Pathology, Odense University, Odense, Denmark and 6Medical Research Council Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK

Received May 7, 2003; Revised July 11, 2003; Accepted July 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. More specifically ADAM12 appeared to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. In the present study we demonstrated that ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. First, we analyzed transgenic mice that overexpress ADAM12 and found mild myopathic changes and accelerated regeneration following acute injury. We then analyzed changes in gene-expression profiles in mdx/ADAM12 transgenic mice compared with their littermate controls and found only a few genes with an expression change greater than 2-fold between mdx/ADAM12 and mdx. The small changes in gene expression were unexpected, considering the marked improvement of the mdx pathology when ADAM12 is overexpressed, and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of {alpha}7B integrin and utrophin, the functional homolog of dystrophin. The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human muscular dystrophies are a group of over 30 different genetic diseases. Many of the muscular dystrophy genes encode proteins that are important for cell adhesion and the structural integrity of muscle. The most common form of muscular dystrophy is the X-linked recessive Duchenne muscular dystrophy. It is caused by mutation(s) in the gene encoding dystrophin and affects ~1/3500 boys (1,2). Other types of muscular dystrophy include laminin {alpha}2-deficient congenital muscular dystrophy (3,4), and the sarcoglycan-deficient limb-girdle muscular dystrophies (5,6). Although our knowledge about the function of the gene products and the consequences of their absence is rapidly advancing, more studies are required to translate this information into new treatment strategies. Genetically modified mouse models are powerful tools to both develop and evaluate the efficiency and risks of new therapeutic targets. The concept of ‘booster genes’ was proposed to denote such gene products that enhance muscle function by providing a functional replacement for the missing gene product(s), and/or by decreasing secondary effects of gene mutation (7).

All adherent cells, including muscle cells, require a physical link between the cytoskeleton and the extracellular matrix. In muscle cells, dystrophin binds to both the internal actin cytoskeleton and the complex of sarcolemmal proteins, in particular dystroglycan, which contributes to the integrity of muscle fibers by binding laminin in the extracellular matrix. In fact, dystrophin deficiency is accompanied by a reduction in dystrophin-associated proteins including dystroglycans and sarcoglycans, resulting in the instability of the sarcolemma and subsequent cell death (2,6,8,9). Several attempts have been made, in both humans and mice, to compensate for the primary dystrophin deficiency by introducing genes into muscle cells. These genes include a full-length dystrophin gene (10), and smaller versions of the dystrophin gene (11), or utrophin, an autosomal homolog that can functionally replace dystrophin (12–14). More recently, other promising approaches have been developed using transgenic technology that consists of enhanced or ectopic expression of gene products, such as {alpha}7 integrin (15) and GalNac transferase (16), that promote survival and/or regeneration of the compromised muscle or affect post-translational modifications of proteins. Although these ‘booster genes’ cannot replace dystrophin, it is evident that they are able to prevent some of the secondary damage caused by dystrophin deficiency, and thus alleviate the muscle pathology. In this context, our recent finding that overexpression of ADAM12 reduces the muscle pathology in mdx mice (17) opens a new avenue for analyzing the functions of cell adhesion proteins and their role in disease.

ADAM12 belongs to a family of transmembrane proteins that includes more than 30 members (18–20). Most members are composed of pro-, metalloprotease, disintegrin-like, cysteine-rich, EGF-like repeat, transmembrane and cytoplasmic tail domains. The ADAMs bear similarity to snake venom metalloproteases or disintegrins (21,22). Disintegrins are a class of peptides isolated from the venom of various snakes. They can interact with certain integrins, and disrupt the binding of the integrin to its ligand. For example by binding to integrin {alpha}IIß3 on platelets, disintegrins prevent platelet aggregation (23). ADAM12 is an active metalloprotease and supports cell adhesion by interacting with syndecan(s) and integrins (24–27). We and others have previously shown that ADAM12 is expressed in skeletal muscle during development and that its expression ceases after birth and reappears in adult muscle during regeneration (17,28,29). In addition, ADAM12 stimulates myoblast cell fusion and myogenesis (28,30). We also generated transgenic mice overexpressing ADAM12 in adult muscle and bred them to the dystrophin-deficient mdx mice. Mdx/ADAM12 mice exhibited significant reductions in muscle necrosis, inflammatory responses, Evans Blue uptake, and serum creatine kinase levels compared with mdx mice (17). This result suggested that ADAM12 reduced or delayed the muscle pathology of mdx mice by preventing muscle cell necrosis, and prompted us to analyze in more detail how ADAM12 influence the normal and the mdx muscle.

In the present study we demonstrate that overexpression of ADAM12 in normal and mdx dystrophic muscle results in increased expression and redistribution of two major adhesion complexes, the {alpha}7ß1 integrin and dystrophin/utrophin-associated glycoproteins. These findings suggest a novel mechanism to compensate for dystrophin deficiency in skeletal muscle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Continuous ADAM12 expression in the normal adult muscle results in mild, non-progressive myopathic changes
We previously reported that ADAM12 alleviates the mdx pathology (17). To determine the possible mechanism, we performed here a more detailed analysis of ADAM12 overexpression on wild-type (Fig. 1) and mdx background. The ADAM12 transgenic mice were fertile, and females delivered and reared normal litters. Transgenic mice were observed for 18 months and they appeared healthy throughout the entire period. In particular, there were no apparent differences between the ADAM12 transgenic mice and their littermate controls with regard to voluntary running or grip strength analysis (data not shown). No apparent pathological changes were observed in hematoxylin and eosin stained sections of hindlimb muscles from 4-day-old and 1-week-old ADAM12 transgenic mice (data not shown). Starting from 2–3 weeks of age, unlike non-transgenic littermate controls, muscles from ADAM12 transgenic mice contained occasional single muscle fibers with segmental necrosis surrounded by inflammatory cells (Fig. 1C and F). More specifically, in sections taken from the middle and distal parts of ADAM12 transgenic mouse tibialis muscles, a total of one to three necrotic fibers were observed (Fig. 1C and F): one to two in the central part (i.e. ~20 per mm3), and zero to one in the distal part (i.e. ~8 per mm3), whereas none were seen in the littermate controls (Fig. 1E and data not shown). The few and discrete segmental necrosis observed in the hindlimb muscles of ADAM12 mice were not extensive compared to the large areas of necrosis seen in mdx dystrophic mice, and serum creatine kinase was not elevated (17) suggesting that overexpression of ADAM12 in the normal muscle was not harmful. Muscle regeneration was evident by the increased number of central nuclei (Fig. 1D and G), which were variably located, but often seen in the proximity of myotendinous junctions. Littermate control mice exhibited only a few fibers with central nuclei. ADAM12 transgenic skeletal muscles m. quadriceps femoris, m. tibialis ant., m. soleus and m. triceps brachii similarly exhibited limited necrosis and regeneration. The diaphragm, in contrast, revealed no necrotic fibers or central nuclei at any age in the more than 100 ADAM12 transgenic mice examined (Fig. 1H and I). No fibrosis, as assessed by van Gieson Hansen staining, was present in muscles from 12- or 18-month-old ADAM12 transgenic mice, compared with littermate controls (data not shown). As described previously (31), adipocytes were present in the perivascular space in 3-week-old mouse muscle, and their numbers gradually increased with age.



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  		Figure 1. ADAM12 transgenic mice with mild myopathic changes. (A) Northern blot of total RNA isolated from ADAM12 transgenic (TG) and littermate control (LC) mouse hindlimb muscles. (B) Immunoprecipitation and blotting of ADAM12 from extracts of transgenic (TG) and littermate control (LC) muscles at the age of 3 weeks, 8 weeks and 6 months. (C–E) Hematoxylin and eosin staining of paraffin sections of m. tibialis from 4-month-old mice: (C) longitudinal section of ADAM12 transgenic muscle showing segmental necrosis; (D) cross-section of ADAM12 transgenic muscle showing central nuclei (arrows); and (E) cross-section of littermate control muscle with peripheral nuclei. Bar, 100 µm. (F) Estimate of the number of necroses per mm3 in the middle part and the distal part of the tibialis ant. of the ADAM12 transgenic mice. No necrosis was observed in the littermate controls (not shown). (G) Estimate of the percentage of central nuclei in the middle part and the distal part of the tibialis ant. of the ADAM12 transgenic mice. Only a few central nuclei were observed in the littermate controls (not shown). (H) Cross-section of diaphragm of ADAM12 transgenic mice and (I) of the littermate controls. Bar, 80 µm.

 
ADAM12 in the normal adult muscle stimulates regeneration following acute injury
Acute freeze injury was induced in the m. tibialis ant. of ADAM12 transgenic mice and non-transgenic littermate controls. Macroscopic inspection of muscle 3 days after injury revealed a grayish spot at the injury site, an indication of necrotic tissue, in control mice (Fig. 2B). In contrast, the muscles of ADAM12 transgenic mice were reddish in color at the injury site, evidence that necrotic tissue had been in part dissolved and replaced by vascularized regenerating tissue (Fig. 2A). Microscopic analysis of muscle 3 days after injury demonstrated extensive necrosis, hemorrhage, and inflammation in the littermate controls (n=14; Fig. 2D and G). In contrast, in the majority of transgenic mice (n=14), numerous basophilic mononuclear cells, most likely representing myoblasts, were already present, indicating an early onset of satellite cell proliferation and differentiation (Fig. 2C) and a higher number of central nuclei was indicative of enhanced regeneration in transgenic muscle (Fig. 2G). Six days after injury, the ADAM12 transgenic mice (n=18) showed less necrosis and fewer inflammatory cells than did the littermate controls (n=13; Fig. 2E–G). Ten days (transgenic n=5, littermate controls n=5) and 21 days (transgenic n=6, littermate control n=4) after injury, tissue integrity was equally well re-established in transgenic and littermate control mice (Fig. 2G and data not shown). Taken together these results suggest that, although the end-point of regeneration following acute injury was the same in ADAM12 transgenic mice and littermate controls, ADAM12 appeared to accelerate the early stages of regeneration.



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  		Figure 2. Accelerated muscle regeneration following acute injury in ADAM12 transgenic mice. M. tibialis was injured by freeze necrosis in ADAM12 transgenic (TG) and normal littermate control (LC) mice. By visual inspection, the injured area was reddish 3 days after injury in ADAM12 transgenic mice (arrows in A), and grayish in the littermate controls (arrows in B). Hematoxylin and eosin staining of paraffin sections of m. tibialis 3 days (C, D) and 6 days (E, F) following injury. Numerous regenerating basophilic mononuclear myoblasts and small blood vessels were seen in the injured area of the ADAM12 transgenic mouse muscle, while extensive necrotic fibers were still seen in the littermate controls. (r) regeneration; (n) necrosis. Bar in (C, D) is 60 µm, and in (E, F) is 30 µm. (G) Necrosis, inflammation (granulocytes, monocytes/macrophages), and regeneration (myoblasts/myotubes) were estimated semi-quantitatively (0 for no changes, 1 for slight changes, 2 for moderate changes, 3 for severe changes as compared to uninjured muscle) and the average presented in the table (*P-value<0.05).

 
The gene expression profile of ADAM12 transgenic mouse muscle
Microarray analysis was performed to reveal ADAM12-induced changes in gene expression. We determined the gene-expression profiles in hindlimb muscles from ADAM12 transgenic, normal littermate controls, mdx, and mdx/ADAM12 transgenic mice. Figure 3 shows the different comparisons and the number of genes that were up- or down-regulated in each combination. Compared with littermate controls (LC), ADAM12 transgenic (TG) mice showed a slight increase in the expression of a broad spectrum of genes related to inflammation, including Mac-2, CD68, osteopontin, cytokine A9, cathepsin S and metallothioneins 1, 2 and 3 (Table 1). Moreover, myogenic factors, myogenin, myoD, and myf-6 showed increased expression in TG compared with LC, whereas expression of myostatin, which inhibits muscle cell proliferation (32), was lower in TG than in LC. Furthermore, the expression of the embryonic myosin heavy chain, which is a marker of newly formed muscle fibers (33), was increased in TG compared to LC. Another apparent embryonic myosin, the cardiac myosin light chain 2 (34), was also increased in TG compared with LC. Other genes encoding muscle structural proteins, such as troponin, tropomyosin, and acetylcholine receptor, were also up-regulated in TG (Table 1). These data corroborate the histological findings showing an increased inflammatory response and enhanced muscle regeneration in TG muscle compared with LC. To confirm the microarray data, northern blot and immunohistochemical staining were performed on a selection of the differentially expressed gene products in the ADAM12 transgenic mouse. Osteopontin mRNA and protein were not detectable in normal hindlimb muscle, but were detected in ADAM12 transgenic muscle (data not shown). The presence of inflammatory cells was confirmed by immunostaining with antibodies to the leukocyte common antigen, CD45 (data not shown) and F4/80 (a macrophage marker; Fig. 4E and F). By immunostaining, myogenin-positive nuclei were detected in the smaller diameter basophilic fibers, confirming an ongoing muscle regeneration (Fig. 4A and B). Neural cell adhesion molecule (NCAM), a cell-surface protein expressed by regenerated muscle fibers (35), was also observed in some muscle cells (Fig. 4C and D).



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  		Figure 3. Gene expression comparison by microarray of ADAM12 transgenic mice and mdx/ADAM12 mice and their respective littermate controls. (A) Phenotypic description of the four types of mice used in this study and the choice of pair wise combinations for gene expression comparison. CK, creatine kinase. (B) The number of genes presenting an expression change higher than 1.5- or 2-fold in each combination.

 

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  		Table 1. Gene expression comparisons of hindlimb muscle from 8-week-old ADAM12 transgenic (TG), mdx/ADAM12 transgenic (mdx/TG), mdx and littermate control (LC) mice
 


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  		Figure 4. ADAM12 transgenic mice express markers of increased muscle regeneration and inflammation. Immunostaining with antibodies against the myogenic factor myogenin (A, B), regeneration marker NCAM (C, D), and macrophage marker F4/80 (E, F) on paraffin sections of hindlimb muscle of a 12-week-old ADAM12 transgenic mouse (TG) muscle (A, C, E) and a littermate control (LC) muscle (B, D, F). Sections are counterstained with hematoxylin. Arrows point to positive immunoreactivity. Bar, 60 µm.

 
Muscle gene expression profile of mdx/ADAM12 transgenic mice
Although the gene expression changes in TG compared to LC mice suggest an increased inflammatory response in transgenic muscle, it is noteworthy that this response was much milder than that observed in mdx mice which have significant inflammation (36). This was confirmed by comparison of gene-expression profiles (Table 1). In fact, compared with LC, mdx muscle showed a dramatic increase in the expression of genes such as osteopontin, CD68 and macrophage metalloelastase. Although the expression of most of these genes was also increased in TG compared with LC, their levels of expression were much lower than in mdx mice (Table 1). Gene expression studies revealed decreased expression of genes involved in the inflammatory response (including Mac-2, osteopontin, and cytokines) in mdx mice with ADAM12 transgene (Table 1). These findings support the previous observation that necrosis is reduced in mdx/ADAM12 mice compared to mdx mice. Most of the genes related to muscle development/regeneration and structure did not show any great difference in expression between mdx/ADAM12 and mdx. In fact, in comparing mdx/ADAM12 and mdx, there were only ten genes that exhibit a difference in expression that is greater than 2-fold (Fig. 3 and in Table 1 marked with a superscript ‘a’). These are: macrophage metalloelastase, osteopontin, small inducible cytokine A2 and A7, myostatin, ankyrin repeat domain 2, cytokeratin, Four and half LIM domain 1, ganglioside-induced differentiation associated protein 1 and S-adenosylmethionine decarboxylase. The minor change in gene expression is surprising considering the dramatic improvement of the mdx pathology with ADAM12 overexpression (17). Thus, we surmised that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally.

Enhanced expression of ADAM12 in the mdx muscle triggers an increased and extrasynaptic localization of {alpha}7 integrin
Defective association of muscle cells with the surrounding basement membrane results in destabilization of the sarcolemma and cell death of the muscle fibers leading to progressive muscle disease (6). In muscular dystrophy this is thought to be caused by deficiency of dystrophin and associated proteins. Muscle pathology in various mouse models has been ameliorated by transgene-mediated expression, or viral-based approaches to specifically replace, or functionally compensate for deficient cell adhesion gene(s) (7,37). We therefore hypothesized that ADAM12, which has been demonstrated to interact with syndecans and integrins (24–27), might influence the expression of muscle cell adhesion protein complexes. The microarray analysis did not detect any major changes in the levels of such transcripts, except for 3-week-old ADAM12 transgenic mice that expressed 1.4-fold more {alpha}7 integrin mRNA than their age-matched, normal littermate controls (data not shown). However, no difference was observed between 8-week-old ADAM12 transgenic mice and their non-transgenic littermates (Table 1). We also confirmed previous data (38) showing that mdx mice expressed more {alpha}7 integrin mRNA than wild-type controls, but we did not detect any further increase in {alpha}7 integrin mRNA in mdx/ADAM12 transgenic mice (Table 1). Based on these observations, we decided to examine the expression of {alpha}7B integrin by immunohistochemistry. Integrin {alpha}7 is enriched at the neuromuscular and myotendinous junction with only minor amounts being present along the lateral parts of the sarcolemma (39). We observed an increase in the intensity of the {alpha}7B integrin immunostaining along the entire sarcolemma in the ADAM12 transgenic mice (Fig. 5B) and in the mdx/ADAM12 mice (Fig. 5F) compared to their respective controls (Fig. 5A and E).The elevated level of {alpha}7B integrin was confirmed by immunoblotting (Fig. 6). When estimated quantitatively, a 2.5-fold increase was observed in the mdx/ADAM12 mice compared with the mdx mice.



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  		Figure 5. Extrasynaptic expression of utrophin and {alpha}7B integrin in ADAM12 transgenic mice. Immunostaining with antibodies against {alpha}7B integrin (A, B, E, F) and utrophin (C, D, G, H) on frozen sections of hindlimb muscle from 8-week-old normal littermate control (LC), ADAM12 transgenic (TG), mdx, and mdx/ADAM12 transgenic (mdx/TG) mice. The expression of both {alpha}7B integrin and utrophin is present in neuromuscular junctions and blood vessels in normal muscle (A, C), whereas in mdx muscle they are present throughout the sarcolemma (E, G) and utrophin appears preferentially in the small fibers (G). In TG and mdx/TG muscle, where ADAM12 is overexpressed, {alpha}7B integrin and utrophin are detected along the sarcolemma of all the muscle fibers (B, D, F, H). Bars, 40 µm.

 


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  		Figure 6. Utrophin and sarcolemmal proteins are upregulated in ADAM12 transgenic mice. Proteins were extracted in a digitonin-containing buffer from the hindlimb muscles of 8-week-old littermate control (LC), ADAM12 transgenic (TG), mdx, and mdx/ADAM12 transgenic mice (mdx/TG) and glycoproteins and associated proteins were enriched using WGA-beads as described in M&M. Equal amounts of proteins of the LC, TG, mdx and mdx/TG muscles were separated on polyacrylamide gels, transferred to nitrocellulose and incubated with antibodies recognizing {alpha}7B integrin; utrophin; {alpha}- and ß-dystroglycans (DG); and {alpha}-, ß- and {gamma}-sarcoglycans (SG). One representative experiment is demonstrated per protein examined, and in the lower right panel the average fold increase in protein expression in at least three immunoblots is demonstrated.

 
Enhanced expression of ADAM12 in mdx muscle triggers increased expression and extrasynaptic localization of utrophin, the functional homolog of dystrophin
In the mdx muscle, utrophin is re-distributed along the entire sarcolemma (40–42) and, when overexpressed, utrophin is able to provide functional compensation for the lack of dystrophin in these mice (12,14). The microarray expression data showed a 1.3-fold increase in the level of utrophin mRNA in the ADAM12 transgenic mice compared to littermate controls, but no increase in mdx/ADAM12 compared to the mdx. Nevertheless, we asked whether the improved pathology observed in the mdx/ADAM12 transgenic mice was associated with altered expression of utrophin protein. Indeed, we observed the presence of utrophin all along the sarcolemma in the muscle of ADAM12 transgenic mice (Fig. 5D) compared with the littermate controls (Fig. 5C). The intensity of the immunostaining was even more increased in mdx/ADAM12 transgenic mice compared with mdx mice (Fig. 5G and H), for which the strongest immunoreactivity was present in the smaller muscle fibers (Fig. 5G). Utrophin exists in two forms, A-utrophin and B-utrophin, that have different expression patterns in the muscle (42). A-utrophin localizes primarily to neuromuscular junctions and peripheral nerves, while B-utrophin is primarily confined to endomysial capillaries and other blood vessels. Moreover, A- but not B-utrophin is up-regulated in the mdx muscle and is distributed along the sarcolemma (42). We used immunohistochemistry to determine which isoform accounts for the utrophin up-regulation that we observed in ADAM12 and mdx/ADAM12 transgenic mice. We found that A-utrophin was enhanced and distributed along the entire sarcolemma of the ADAM12 and mdx/ADAM12 muscle, whereas the expression and localization of B-utrophin was unchanged (data not shown). Finally, we examined whether the extrasynaptic localization of utrophin was due to an increased amount of this protein. Indeed, immunoblot experiments showed higher amounts of utrophin in ADAM12 transgenic mice (Fig. 6), and a comparison of the intensity of immunoblot bands demonstrated a 1.8-fold increase in mdx/ADAM12 transgenic compared with the mdx mice muscle. As the microarray expression data did not show any increase in utrophin mRNA level in the mdx/ADAM12 muscle, a post-transcriptional regulation of utrophin is likely, as previously described (42–44).

Enhanced expression of ADAM12 in mdx muscle triggers an increased expression of dystrophin-associated glycoproteins
One of the major causes of pathology of the mdx dystrophin-deficient muscle is the secondary deficiency of dystrophin-associated proteins (6). To test if the increase of utrophin in ADAM12 transgenic mice restores the expression of dystrophin-associated proteins, we used immunoblotting to assess the expression levels of several proteins belonging to this complex. We found an increase in {alpha}- and ß-dystroglycan in ADAM12 and mdx/ADAM12 transgenic mice compared with their littermate controls (Fig. 6). Similar results were obtained with two different monoclonal antibodies to {alpha}-dystroglycan, the IIH6 (45) (Fig. 6) and VIA4–1 (46) (data not shown). The levels of {alpha}-, ß- and {gamma}-sarcoglycans were also slightly increased (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We demonstrate here that overexpression of ADAM12 induces increased expression and redistribution of two cell adhesion receptor complexes, {alpha}7 integrin and the dystrophin/utrophin-associated glycoproteins in the normal adult muscle as well as in the mdx dystrophic muscle. These results strongly suggest that ADAM12 can directly or indirectly influence cell membrane composition in the muscle, and thereby contribute to membrane integrity and muscle cell stability. These results add to our understanding of mechanism of disease in muscular dystrophy and the role of ADAM12 in muscle cell biology and pathology. Furthermore, the finding of significant ADAM12-induced up-regulation of both {alpha}7B integrin and utrophin along the extrasynaptic sarcolemma places ADAM12 activities on the list of potential ‘booster genes’, which may point to novel directions for the development of pharmacological strategies for treating various forms of muscular dystrophy (7,37).

Muscle cells possess two major adhesion complexes that connect the intracellular actin cytoskeleton to the basement membrane, {alpha}7ß1 integrin and the dystrophin/utrophin-associated glycoproteins (2,47). Although the exact role(s) of these adhesion complexes in the muscle are unknown, their absence has a dramatic effect on the integrity of the muscle cell membrane, i.e. muscle cells undergo apoptosis and necrosis accompanied by inflammation and regeneration. The end result of cycles of degeneration and regeneration is the loss of functional muscle mass and replacement of muscle cells with connective tissue and fat. Elevated creatine kinase levels in serum indicate muscle damage, and combined with morphological changes, are diagnostic of ongoing muscular disease (48). Humans with Duchenne muscular dystrophy and mdx mice carry X-linked recessive mutations in the gene encoding dystrophin, a large membrane-associated cytoskeletal protein located just beneath the sarcolemma membrane. The lack of dystrophin also results in a secondary loss or reduced expression of dystrophin-associated glycoproteins including the dystroglycans and the sarcoglycans (9). Similarly, mutations in the gene encoding {alpha}7 integrin result in myopathic changes (49), and {alpha}7 integrin is reduced in several myopathies of unknown etiology (50). Conversely, transgenic overexpression of either utrophin (12,13) or {alpha}7 integrin (15) can compensate for the lack of dystrophin in mdx mice and bring about structural and functional improvement in pathology. Thus, utrophin can replace dystrophin, restore the expression of dystrophin-associated glycoproteins (DAGs) in the mdx mouse and thereby directly prevent or delay the onset of pathology (51). The mechanism by which {alpha}7 integrin alleviates the mdx pathology seems to be different (15). A 2-fold increase in {alpha}7 integrin in mdx/utr-/- mice had a strikingly positive effect on the disease; however, enhanced {alpha}7ß1 expression did not prevent the initial phases of degeneration, but rather provided stabilization after the subsequent regeneration (15). Since an {alpha}7 integrin-induced substitution of dystrophin with utrophin is not possible in the mdx/utr-/- mice, this study provided direct evidence for a specific role of the {alpha}7ß1 integrin adhesion complex in maintaining integrity of the muscle (15). In the present study we provide evidence for the concept that ADAM12 expression in adult mdx mouse muscle, by increasing or redistributing the expression of both the {alpha}7 integrin adhesion complex and the DAG adhesion complex may prevent the initial muscle cell necrosis in the mdx mice. Further studies are needed to explore whether ADAM12 may also influence other cell adhesion molecules, such as the cadherins (52). Although ADAM12 may prevent necrosis in mdx muscle, it seems a paradox that it induces mild myopathic changes in the normal hindlimb muscle. The use of the muscle creatine kinase (MCK) promoter results in lower expression of ADAM12 in diaphragm than in hindlimb muscle of transgenic mice (17), and it is noteworthy that ADAM12 did not induce necrosis in normal diaphragm. Furthermore, in other lines of ADAM12 transgenic mice with fewer copies of the transgene, we observed less necrosis in hindlimb muscle (data not shown). Given that a protein may have different effects depending on its cellular environment, overexpression of ADAM12 may be slightly toxic in normal muscle (depending on expression level), but not in dystrophin-deficient muscle.

We found that ADAM12 increased the level of utrophin expression and induced its localization along the sarcolemma in ADAM12 transgenic mice. Utrophin is a large 395 kDa cytoskeletal protein that is expressed along the entire sarcolemma during muscle development and is restricted to the neuromuscular junction after birth (40,53,54). In certain diseases, including Duchenne muscular dystrophy and polymyositis, the expression of utrophin is also increased and present along the extrasynaptic sarcolemma of muscle fibers (55,56). Utrophin exists in two variants, A- and B-utrophin, which are transcribed from different promoters. In normal adult skeletal muscle, A-utrophin is expressed at the neuromuscular junction, while B-utrophin is expressed in the blood vessels. Only A-utrophin is up-regulated and redistributed throughout the entire sarcolemma of the mdx mouse muscle fibers (42). In ADAM12 transgenic mice we observed that A-utrophin, but not B-utrophin is up-regulated in comparison to non-transgenic controls. Similarly, in mdx/ADAM12 mice, A-utrophin was up-regulated to a significantly greater degree than it was in the mdx mice. In our DNA array analysis we observed a 1.3-fold increase in the level of utrophin mRNA in ADAM12 transgenic mice compared with littermate controls, but no changes were observed in mdx/ADAM12 mice, as compared with mdx mice. Studies on the regulatory events controlling the expression and localization of utrophin at the neuromuscular junction suggest that both local transcriptional activation as well as post-transcriptional mechanisms are important (44). It is, therefore, logical to suggest that ADAM12 may, directly or indirectly, influence the targeting and stability of utrophin mRNA and/or protein in muscle.

ADAM12 activities are potential targets to consider in the search for pharmacological interventions directed at up-regulating the expression of adhesion receptors or utrophin along the extrasynaptic sarcolemma of the dystrophic muscle (7,37). However, in designing such therapies it will be essential to address key questions such as dose and possible side-effects. Some genes of the adhesion complexes may not efficiently compensate for dystrophin deficiency, and some may even be toxic to the muscle if overexpressed. For example, mice that overexpressed {gamma}-sarcoglycan under the muscle-specific creatine kinase promoter developed a severe muscular dystrophy (57). Even if {alpha}- and ß-sarcoglycan were up-regulated at the cell membrane, {gamma}-sarcoglycan failed to reach the cell surface and was associated with intracellular aggregates. We found that overexpression of ADAM12 in transgenic mice produced mild non-progressive myopathic changes with a few scattered necroses and central nuclei indicating ongoing remodeling. No fibrosis developed, even after 1 year of observation, and the mice did not develop any apparent dysfunction. To determine the relative contribution of the intracellular versus extracellular part of ADAM12, we have recently developed transgenic mice that overexpress a membrane-anchored form of ADAM12 without the cytoplasmic tail (ADAM12-{Delta}cyt). These ADAM12-{Delta}cyt mice developed similar mild myopathic changes, and expression of the ADAM12-{Delta}cyt transgene also induced the increased extrasynaptic expression of utrophin and significantly improved the pathology of mdx mice (data not shown). We therefore propose that the intriguing activities of ADAM12 at the sarcolemma that were revealed in these studies lie in the extracellular region of this protein. In this context, it is also important to note that overexpression of the non-membrane-anchored, shorter and secreted form of ADAM12 (ADAM12-S) did not result in any signs of muscle necrosis or central nuclei (31).

ADAM12 overexpression upregulates {alpha}7 integrin, utrophin, and dystrophin-associated proteins, and redistributes {alpha}7 integrin and utrophin into extrasynaptic locations, thereby potentially influencing the assembly of functional cell adhesion complexes at the sarcolemma. The molecular mechanisms underlying these effects are not known, but a number of models can be considered. One is that ADAM12, through its cell-adhesion and metalloprotease domains, associates with and influences the function of other cell-surface proteins, including integrins, syndecans and growth factors. Another model is that ADAM12 keeps the muscle in an immature mode. ADAM12 is expressed during development and its expression is down-regulated in early postnatal life as the muscle matures. ADAM12 is normally expressed during regeneration, but only in myoblasts and newly formed myotubes. Thus it is a common feature of development and regeneration that ADAM12 disappears as the myotube matures. This suggests that disappearance of ADAM12 may be required for full maturation of muscle, and maturation may be inhibited by continuous expression of ADAM12. It is possible that only fully mature muscle is dependent on dystrophin, while immature muscle may have enough utrophin in appropriate locations. It will be interesting to further examine how a muscle in an apparent immature state, as defined by the persistent expression of ADAM12 and the extrasynaptic expression of {alpha}7 integrin and utrophin, is more resistant to the development of dystrophin-deficient muscular dystrophy and regenerates faster upon acute injury.

In conclusion, the results described here highlight the beneficial role of ADAM12 in the dystrophin deficient muscle, and provide a novel paradigm for achieving compensation for dystrophin deficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
ADAM12 transgenic and dystrophin-deficient mdx mice
Transgenic mice expressing full-length human ADAM12-L cDNA (GenBank AF023476) under the control of the MCK promoter were generated as described (17). Transgenic founders were obtained on the C57BL/6JxCBA, F1 background and these were bred into this or the C57BL/6J strain. Homozygous female mdx mice (C57BL/10ScSn-Dmd<mdx>/J) were obtained from Jackson laboratories and mated with male ADAM12 transgenic mice as described (17). The presence of the human ADAM12 transgene was determined by PCR analysis of genomic DNA isolated from tail biopsies. Dystrophin deficiency was confirmed by immunohistochemistry. Northern blots were performed as previously described (58) to confirm the presence of ADAM12 transcripts. Two tests were performed to evaluate the overall muscle function: running exercise, and the measurement of grip strength. Mice were allowed to exercise voluntarily for an average of 2 weeks in cages containing exercise wheels (35 cm diameter) equipped with counters to register the number of wheel revolutions (Ugo Basile, Comerio, Italy). In separate tests the grip strength was determined with an electronic pull strain gauge that was fitted directly to the grasping wire (Ugo Basile, Comerio, Italy). All experiments were conducted according to the animal experimental guidelines of the Animal Inspectorate, Denmark.

Muscle regeneration
Regeneration of skeletal muscle was induced by the freeze–crush injury of m. tibialis anterior (59). Briefly, mice were anesthetized and following a skin incision the muscle was exposed. The injury was induced using a metal rod (2.5 mm in diameter) cooled in liquid nitrogen that was then gently pressed against the exposed muscle for 5 s. The skin was sutured and the mice were sacrificed 3, 6, 10 and 21 days following the time of injury. A total of 43 ADAM12 transgenic mice and 36 littermate controls were used. The injured and control muscles were removed, processed for histological analysis, and necrotic fibers, inflammatory cell infiltration (granulocytes and monocytes/macrophages) and myoblasts/myotubes were estimated semi-quantitatively (0 for no changes, 1 for slight changes, 2 for moderate changes, 3 for severe changes compared with uninjured muscle), the average calculated, and statistical analysis performed using the Student's t-test. A P-value of <0.05 was considered statistically significant.

Histological analysis and immunohistochemical staining
Muscle tissue specimens from mice at various ages were fixed in formalin and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin or, in selected cases, with Van Gieson Hansen. Sections from the middle and distal parts of isolated m. quadriceps femoris, m. tibialis ant., m. soleus and m. triceps brachii were analyzed to morphologically estimate the proportion of the section that contained necrotic fibers surrounded by mononuclear inflammatory cells or central nuclei. For immunohistochemical staining on paraffin sections, the following monoclonal mouse antibodies were used: myogenin (F5D, DAKO, Glostrup, Denmark) at 1 : 200; rat anti-mouse macrophage antibody F4/80 (Serotec, Oxford, UK) at 1 : 800; rat anti-mouse CD45 (SRT5, Serotec) at 1 : 100; and a polyclonal rabbit anti-NCAM (kindly provided by E. Bock, University of Copenhagen) at 1 : 800. The sections were pretreated for antigen retrieval by either protease pretreatment or by heating in a microwave oven. For immunostaining on frozen sections, the following polyclonal rabbit antibodies were used: rb 122 (1 : 100) that recognizes the cysteine-rich domain of ADAM12 (17); affinity-purified anti-{alpha}7B integrin (10 µg/ml) (60); affinity-purified antibodies to a synthetic peptide from the 13 most C-terminal amino acids of utrophin (10 µg/ml); and anti-A- and B-utrophin (10 µg/ml) (42). The sections were incubated with primary antibodies for 1 h at room temperature. Detection was performed with FITC-conjugated goat anti-rabbit IgG (Jackson Immunolab) or with the DakoChem Mate detection kit (code K 5001), which is based on an indirect streptavidin–biotin technique with a biotinylated secondary antibody.

Immunoprecipitation and immunoblotting
To determine the amount of ADAM12 protein expressed in muscle, muscle tissue extracts were homogenized in TBS (20 mM Tris–HCl, pH 7.4 and 140 mM NaCl) containing protease inhibitors (Complete, ethylenediaminetetraacetic acid-free protease inhibitor cocktail tablets; Roche Molecular Biochemical, Hvidovre, Denmark). After the addition of equal volume of RIPA buffer (1% Triton X-100, 25 mM HEPES, 150 mM NaCl, 0.2% SDS, 1% deoxycholate, 5 mM MgCl2, 50 mM Tris–HCl pH 7.4) and centrifugation to remove debris, cleared supernatants were used for immunoprecipitation with mouse monoclonal antibodies (8F8, 6E6 and 6C10) (17) followed by SDS–PAGE and immunoblotting with a polyclonal antiserum to a C-terminal peptide of ADAM12 (rb 109) (17). To examine the expression of components of muscle cell adhesion proteins, 100 mg samples of hindlimb muscle tissue were dissected from ADAM12 transgenic mice, littermate controls, mdx mice and mdx/ADAM12 mice. Each tissue sample was homogenized in 2 ml extraction buffer (50 mM Tris–HCl, pH 7.4, 500 mM NaCl, 1% digitonin, 600 ng/ml pepstatin A, 500 ng/ml aprotinin, 500 ng/ml leupeptin, 100 µM phenylmethyl sulfonyl fluoride, 750 µM benzamidine, 760 ng/ml calpain inhibitor I, and 720 ng/ml calpeptin) with an Ultra-Turrax (T25; Ika, Stauffen, Germany), then further processed using a dounce homogenizer (Potter S; Braun Biotech International, Melsungen, Germany). Homogenates were incubated in an end-over-end mixer for 1 h at 4°C, followed by centrifugation for 30 min at 145 000g in an ultra-centrifuge (Sorvall M120SE; Kendro, Hanau, Germany). The protein concentration of the supernatants was estimated by BCA protein assay kit (Pierce, Rockford, USA), and each of the extracts (8 mg total protein) was incubated overnight at 4°C with 200 µl wheat germ agglutinin-beads (WGA-beads; Vector, Burlington, USA) in an end-over-end mixer. The beads were washed three times with TBS +0.1% digitonin, resuspended in 400 µl sample buffer, and boiled. Aliquots (50 µl) were then separated on a 3–15% polyacrylamide gel, and separated proteins were transferred to nitrocellulose membranes. Loading of equal amounts of protein was confirmed by red ponceau staining of the membranes. The following primary antibodies were used to immunostain the membranes: affinity-purified anti-{alpha}7B integrin (10 µg/ml) (60), anti-{alpha}-dystroglycan (IIH6C4 and VIA4-1; Upstate, Charlottesville, VA, USA), anti-ß-dystroglycan (NCL-ß-DG; Novocastra, Newcastle upon Tyne, UK), anti-{alpha}-sarcoglycan (NCL-{alpha}-SG; Novocastra, Newcastle upon Tyne, UK), anti-ß-sarcoglycan (NCL-ß-SG; Novocastra, Newcastle upon Tyne, UK), anti-{gamma}-sarcoglycan (NCL-{gamma}-SG; Novocastra, Newcastle upon Tyne, UK), and anti-utrophin peptide antiserum (see above). The following horseradish peroxidase-linked secondary antibodies were used: sheep anti-mouse IgM (115-036-075; Jackson Laboratories, Bar Harbor, ME, USA) and sheep anti-mouse Ig (Amersham, Piscataway, NJ, USA). Detection was performed using the ECL system (RPN2106; Amersham, Piscataway, NJ, USA). At least three immunoblots were performed for each protein and the average intensity of stained bands was calculated using the public domain NIH Image program.

Gene expression and DNA microarray analysis
Quadriceps muscle from 10 mice (8 weeks old) of each type (ADAM12 transgenic, littermate control, mdx and mdx/ADAM12) were pooled into two replicate samples, each containing muscles from five mice. Total RNA was isolated from each pool using TRIzol reagent (Invitrogen, Tåstrup, Denmark) and further purified with RNeasy® kit (Qiagen, Albertslund, Denmark). Eight micrograms of purified RNA were used to synthesize double-stranded cDNA using Superscript® Choice System (Invitrogen, Tåstrup, Denmark) with an oligo-dT primer containing a T7 RNA polymerase promoter (GenSet, Evry, France). The cDNA was used as a template for an in vitro transcription reaction to synthesize biotin-labeled antisense cRNA (BioArrayTM High Yield RNA Transcript Labeling Kit; Enzo Diagnostics, Farmingdale). After fragmentation at 94°C for 35 min in fragmentation buffer (40 mM Tris, 30 mM MgOAc, 10 mM KOAc), the labeled cRNA was hybridized for 16 h to Affymetrix MG-U74Av2 arrays (Affymetrix Inc., Santa Clara, CA, USA), which contain ~12 000 probe sets corresponding to 6000 ESTs and 6000 annotated genes. The arrays were washed and stained with phycoerytrin streptavidin (SAPE) using the Affymetrix Fluidics Station® 400. The arrays were scanned in the Affymetrix GeneArray® 2500 scanner, as described in the Affymetrix GeneChip® protocol, to generate fluorescent images. Low-level analysis was done using MicroArray Suite, version 5 software from Affymetrix. This basic analysis includes an absolute, and a comparison analysis (61). In the absolute analysis a detection call and an intensity value (expression level) of each gene represented on the array is calculated based on the hybridization signal from 16 perfect match and mismatch probe pairs. The detection call is assigned a value of ‘present’, ‘marginal’ or ‘absent’ based on whether the transcript is determined to be present or absent in the sample. In the comparison analysis, two microarrays (an experimental and a baseline sample) are compared and the intensity value difference and a change call, indicating whether a gene has an increased or decreased expression level compared with the baseline sample, is calculated and coupled with a signal log ratio (SLR). The SLR is a quantitative measure of the differential expression given as the log2 of the fold change ratio (61). For all the conditions compared, both replicates were used to generate four pair-wise comparisons. Genes that showed a SLR value greater than 0.6 or 1 (increased more than 1.5- or 2-fold, respectively) or SLR value smaller than -0.6 or -1 (decreased more than 1.5- or 2-fold) in all four pair-wise comparisons were selected for further analysis, as previously described (62).


   ACKNOWLEDGEMENTS
 
We thank Brit Valentin for technical assistance, Bent Børgesen for photographic assistance, Per Dalgaard for artwork and Maryellen Daston for editorial assistance. The study was supported by grants from the Danish Medical Research Council, Neye Foundation, the Danish Cancer Society, the Velux, Novo Nordisk, Munksholm, Friis, Toyota and Haensch Foundations (U.W.); by the Danish Institute of Neuromuscular Diseases, Mads Clausen's Fund, and the Danish Stem Cell Center (H.D.S.), by an EU grant, Quality of Life and Management of Living Resources [contract no. QLG1-CT-1999-00870, designated Genetic Resolution of Myopathies: European cluster (Myocluster)] (U.W. and T.V.), by the National Institutes of Health (E.E.), the Muscular Dystrophy Association USA (K.D., E.E. and U.W.), by the Deutsche Forschungsgemeinschaft (Str 498/3-2; T.V.) and Muscular Dystrophy Campaign UK (K.D.). B.M. is supported by a Marie Curie fellowship (contract no. HPMF-CT-2002-01557) and N.K. by the Japan Society for the Promotion of Science.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Institute of Molecular Pathology, University of Copenhagen, Frederik V's vej 11, 2100 Copenhagen, Denmark. Tel: +45 3532 6056; Fax: +45 35326081; Email: ullaw{at}pai.ku.dk Back


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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