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Am J Physiol Cell Physiol 292: C2141-C2149, 2007. First published January 10, 2007; doi:10.1152/ajpcell.00295.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

beta-Defensin overexpression induces progressive muscle degeneration in mice

Yasuhiro Yamaguchi,1,3,4 Takahide Nagase,2 Tetsuji Tomita,1 Kyoko Nakamura,4 Shigetomo Fukuhara,4 Tomokazu Amano,3 Hiroshi Yamamoto,1 Yukie Ide,5 Misao Suzuki,6 Shinji Teramoto,1 Tomoichiro Asano,3 Kenji Kangawa,7 Naomi Nakagata,5 Yasuyoshi Ouchi,1 and Hiroki Kurihara3,4

Departments of 1Geriatric Medicine, 2Respiratory Medicine, and 3Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo; 4Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, and Divisions of 5Reproductive Engineering and 6Transgenic Technology, Center for Animal Resources and Development, Kumamoto University, Kumamoto-shi, Kumamoto; and 7Department of Biochemistry, National Cardiovascular Center Research Institute, Suita-ku, Osaka, Japan

Submitted 27 May 2006 ; accepted in final form 4 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse beta-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed I{kappa}B{alpha} accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.

muscular dystrophy; innate immunity; NF-{kappa}B

ANTIMICROBIAL PEPTIDES have emerged as a part of the host defense mechanism in animals and plants (16, 29). Defensins comprise a family of mammalian cationic antimicrobial peptides, and {alpha}-, beta-, and {theta}-defensin subfamily members exist that conserve three specific disulfide pairings. They are produced by leukocytes and various types of epithelial cells constitutively or in response to microbial signals and inflammatory cytokines (4, 9, 10, 14, 15).

While the immune response is indispensable for the survival of humans, the chronic inflammatory response is harmful and leads to various common disorders. In addition to the potent antimicrobial effects, defensins could act on diverse immune cells and epithelial cells through CCR6, Toll-like receptor-4 (TLR4), or other mechanisms, regulating the whole immune response (5, 23, 26, 37, 40, 41). Furthermore, they exert cytotoxic effects on mammalian cells themselves (18). {alpha}-Defensin causes cell lysis of variable cultured cells through the permeabilization of cell membrane (19) and subsequent DNA injury (11, 18). Although little information had been reported regarding beta-defensin cytotoxicity (21, 23), treatment of mouse blastocysts with human beta-defensin-2 (hBD-2) led to their degeneration and death (28). So some participation of the defensin family would be likely in the pathogenic immune response of various diseases (32).

Recently, we identified mouse beta-defensin-6 (mBD-6), a beta-defensin subfamily member expressed in skeletal muscle (39). mBD-6 expression was also augmented by bacterial endotoxin, perhaps under the regulation of the NF-{kappa}B pathway like hBD-2 and mouse beta-defensin-3 (3). To explore the novel effects of this molecule, we generated transgenic mice overexpressing mBD-6 constitutively. Here, we show that the dysregulated beta-defensin expression resulted in extensive myofiber degeneration, reminiscent of human muscular dystrophy.

Muscular dystrophy is an inherited disorder characterized by progressive muscle degeneration. The most common form, Duchenne muscular dystrophy, is caused by mutations in the dystrophin gene, and other causative molecules like dystroglycan and sarcoglycan organize dystrophin-glycoprotein complex binding to laminin (6, 17, 30). Another form of muscular dystrophy, limb-girdle muscular dystrophy type 2A (LGMD2A), has proved to be due to the defects of calpain-3, a proteolytic enzyme (24, 31, 35). While the identification of responsible genes for muscular dystrophy has improved, the pathogenic mechanisms are not clear enough to date. Multiple factors, including immune response, are related to the pathophysiology of muscular dystrophy (33). The present finding may give a clue to the novel involvement of innate immunity in degenerative diseases like muscular dystrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of transgenic mouse. The transgene was constructed by inserting the mBD-6 cDNA 3' downstream of the chicken beta-actin promoter in the pCAGGS plasmid (a gift from J. Miyazaki) (22). The excised transgene was microinjected into fertilized C57BL6/J mouse oocytes. The genomic DNA was isolated from the mice tail and analyzed by Southern blotting and/or PCR. On Southern blot analysis, the genomic DNA was digested with BglII, resolved on 1.0% agarose gel, and transferred to the membrane. A 250-bp fragment containing the second exon of mBD-6 was labeled by [32P]dCTP using a random primed DNA labeling kit (Roche). The labeled probe was hybridized to the membrane using ExpresHyb (Clontech). PCR was performed using the primers spanning mBD-6 cDNA (forward primer, 5'-GGTTATTGTGCTGTCTCATC-3'; reverse primer, 5'-ATTTGTGAGCCAGGGCATTG-3'). The PCR condition was 94°C for 40 s, 60°C for 30 s, and 72°C for 60 s, carried out for 35 cycles. The one line, Tg(CAG-mBD6)1 mice, was maintained on ICR mice, and the other line, Tg(CAG-mBD6)2 mice, was maintained on C57BL6/J mice. The immunohistochemical analysis was performed on Tg(CAG-mBD6)1 mice after they were backcrossed to the C57BL6/J strain. Animal care and use in our laboratory were in strict accordance with the guidelines for animal and recombinant DNA experiments put forth by Kumamoto University and The University of Tokyo. The experiment was approved by the Committee for Animal Resources in Kumamoto University and by the University of Tokyo.

RT-PCR. Total RNA was isolated from the skeletal muscle using ISOGEN (Nippon gene). Five micrograms of each sample were reverse-transcribed using SuperscriptII (Gibco-BRL). To detect the expression of transgene, we designed a forward primer from the mBD-6 first exon (5'-ACCATGAAGATCCATTACCTG-3') and a reverse primer from the rabbit beta-globin (5'-ATTTGTGAGCCAGGGCATTG-3'), and we performed a real-time PCR reaction using the Fluorescent Quantitative Detection System Version 3.02 (LineGene).

Antiserum preparation. The putative mature peptide composed of the COOH-terminal 40 amino acids of mBD-6 was chemically synthesized at the Peptide Institute (Minoh, Japan), as previously described (39). The anti-mBD-6 rabbit serum was prepared at the Peptide Institute using this synthetic mBD-6 peptide, conjugated to keyhole limpet hemocyanin, and injected into rabbits.

Isolation of mBD-6 peptide. We followed the procedure described by Valore et al. (36). Frozen skeletal muscle was homogenized in ISOGEN (Nippon gene), and the protein was extracted according to the manufacturer's protocol. Protein pellets were incubated overnight in 5% acetic acid at 4°C, and the dissolved proteins were neutralized with 10% NH3. These protein solutions were separated on Tris-glycine SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The membrane was probed with anti-mBD-6 rabbit serum, followed by a peroxidase-conjugated anti-rabbit IgG antibody (ICN), and visualized using the Enhanced Chemiluminescence Plus System (Amersham Pharmacia Biotech).

Evaluation of muscle strength. We evaluated muscle strength by measuring the time during which mice could hang down from a stainless steel lattice. The procedure was repeated twice for each mouse, and the better record was indicated.

Evans blue dye staining and measurement of serum creatine kinase activity. We performed the intraperitoneal injection of 10 mg/ml Evans blue dye solution of phosphate-buffered saline (PBS) (0.1 ml/10 g body wt) on 2-mo-old Tg1 mice and wild-type mice. The skeletal muscle samples were removed 16 h after the injection. The frozen 10-µm sections were fixed in acetone for 1 min and observed under a fluorescence microscope. Serum creatine kinase (CK) activity was measured at SRL (Tachikawa, Japan), a commercial laboratory.

Tissue preparation and immunohistochemistry. Skeletal muscle samples were removed and frozen in isopentane chilled in liquid nitrogen. The frozen 10-µm sections were processed for hematoxylin and eosin staining or immunohistochemical analysis. For the immunohistochemistry, the sections were fixed in acetone for 5 min and probed with the following primary antibodies: anti-dystrophin for COOH terminus (C-20) (Santa Cruz), anti-{alpha}-dystroglycan for internal core part (E-21) (Santa Cruz), anti-laminin-{alpha}2 chain (LSL), anti-neural cell adhesion molecule (Chemicon), anti-I{kappa}B{alpha} (Santa Cruz), anti-cleaved-caspase-3 (Trevigen), and anti-calpain-3 antibody (a gift from H. Sorimachi).

Statistics. Comparison of the body weights or serum CK activity was made with Student's t-test. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation of transgenic mice overexpressing mBD-6. To achieve broad and high expression of mBD-6, a cDNA fragment encoding mBD-6 was connected to the 3'-end of the chicken beta-actin promoter flanked with a cytomegalovirus immediate-early enhancer (Fig. 1A). While six founder transgenic mice were identified using PCR and Southern blot analysis, the transgene was transmitted to germline in two lines. On Southern blot analysis, the one line, Tg(CAG-mBD6)1, was estimated to harbor several copies of the transgenes because of multiple extra bands, including a 2.0-kb DNA fragment corresponding to the full-length transgene size, while the wildtype genomic DNA showed two copies of the 1.4-kb intrinsic mBD-6 gene and a more faint 3.2-kb band perhaps composed of the mBD-6 pseudogene. The other line, Tg(CAG-mBD6)2, was estimated to harbor a single copy because of the single 3.4-kb extra band (Fig. 1B). Tg(CAG-mBD6)1 and Tg(CAG-mBD6)2 mice will be referred to as Tg1 mice and Tg2 mice, respectively.


Figure 1
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  		Fig. 1. Mouse beta-defensin-6 (mBD-6) transgene expression. A: schematic description of the mBD-6 transgene fragment used to generate transgenic mice. A human cytomegalovirus immediate-early (CMV-IE) enhancer is linked to the chicken beta-actin promoter, followed by its first exon and intron. In addition, a rabbit beta-globin poly (A) sequence is located downstream from the mBD-6 cDNA. Black bar indicates probe of second exon of mBD-6 for Southern blot analysis in B. Arrows indicate the primers for RT-PCR of mBD-6 transgene. B: Southern blot analysis of the BglII-digested genomic DNA from Tg(CAGmBD6)1 mice (Tg1) and Tg(CAGmBD6)2 mice (Tg2). Wildtype genomic DNA showed two copies of the 1.4-kb intrinsic mBD-6 gene, and the more faint 3.2-kb band is perhaps composed of the mBD-6 pseudogene. Tg1 showed multiple extra bands, including a 2.1-kB DNA fragment corresponding to the full-length transgene size, while Tg2 showed a single 3.4-kb extra band. C: RT-PCR of mBD-6 transgene mRNA. The transgene-specific RT-PCR indicated transgene expression in skeletal muscle of Tg1 and Tg2. G3PDH, glyceraldehyde-3-phosphate dehydrogenase. D: Western blot analysis of mBD-6 peptide extracted from skeletal muscle; 280 ng of synthetic mBD-6 peptide composed of 40 NH2-terminal residues were used as standard. mBD-6 peptide was detected in the extracts from Tg1 and Tg2 skeletal muscle but not from the wild-type mice (WT).

 
In both lines, transgene expression was detected in the skeletal muscle by RT-PCR using the primers from mBD-6 cDNA and rabbit beta-globin cDNA (Fig. 1, A and B). Tg1 mice showed 3.4 times higher expression of the transgene than Tg2 mice. Western blot analysis could detect mBD-6 peptide in the extracts from Tg1 and Tg2 skeletal muscle, and the expression level was also low in Tg2 mice (Fig. 1C). In wild-type mice, the mBD-6 signal was not detected by Western blot analysis under the same experimental condition. Immunohistochemical analysis of the mBD-6 peptide showed the mBD-6 peptide stored in the cytoplasmic granules in some skeletal muscle myofibers of Tg1 mice.

Tg(CAG-mBD6) mice develop muscle degeneration. At birth, Tg1 and Tg2 mice were indistinguishable from their wild-type littermates. By 6 wk of age, poor growth of Tg1 mice became evident, and at 8 wk of age, both the male and female body weights of Tg1 mice were significantly lower than those of their wild-type littermates (P < 0.01) (Fig. 2A). The mean body weight of Tg1 mice was ~80% that of their wildtype littermates. They showed contracted stiff limbs and progressive kyphosis by 6 mo of age (Fig. 2B). Most of Tg1 mice died before 11 mo of age. The short lifespan of Tg1 mice was more evident after they were backcrossed to the C57BL6/J strain. Many of the Tg1 mice died before 8 mo, and no mice backcrossed to the C57BL6/J strain lived for >1 yr. We could not clarify the specific cause of Tg1 mouse death except severe loss of body weight. Although the other transgenic line, Tg2, did not reveal so prominently poor growth or kyphosis, 6-mo-old Tg2 male mice also showed significantly lower body weights than wild-type littermates. Tg2 mice lived for >12 mo.


Figure 2
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  		Fig. 2. Poor growth and progressive kyphosis of Tg1 mice. A: comparison of body weights between Tg and WT mice. Each circular point (bullet) indicates the measured individual body weight. Mean values ({blacklozenge}) ± SD are also shown. Both the male and female body weights of Tg mice were significantly lower than those of WT. B: photograph of 6-mo-old Tg1 mouse. Arrow indicates kyphosis.

 
Regarding the decreased body weight, we evaluated the food intake of 5-wk-old Tg1 mice. The food intake of Tg1 mice was 3.31 ± 0.12 g/day, which was significantly lower than the food intake of the wild-type littermates, which was 4.00 ± 0.29 g/day (P < 0.05).

Because progressive kyphosis is a prominent feature caused by functional impairment of skeletal muscle, we evaluated the muscle strength of Tg1 mice by hanging them from a stainless steel lattice. Two-month-old Tg1 mice dropped in a significantly shorter time from the lattice, indicating muscle weakness in Tg1 mice. While most of the wild-type littermates hung for >120 s, significantly more Tg1 mice dropped before 60 s (P < 0.01) (Fig. 3).


Figure 3
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  		Fig. 3. Evaluation of muscle strength of Tg1 mice. We evaluated muscle strength by measuring the time during which mice could hang down from a stainless steel lattice. Graph shows percentage of mice that could hang down for the indicated time. While most of the WT littermates hung down for >120 s, many of Tg1 mice dropped before 60 s.

 
Hematoxylin and eosin staining of skeletal muscle revealed degenerative myofibers, infiltration of mononuclear cells, and some centronucleated myofibers in 4-wk-old Tg1 mice. After 8 wk of age, centronucleated myofibers became much more predominant, and faintly stained necrotic myofibers, basophilic regenerating myofibers, and fiber splitting were frequently encountered. The myofibers also showed prominent differences in size (Fig. 3). These features were observed in all skeletal muscles examined, including gastrocnemius muscles, anterior tibial muscles, soleus muscles, diaphragm, and muscles of the back. Meanwhile, no histological abnormalities were noted in 20-day-old Tg1 mice (Fig. 4). The other transgenic line, Tg2, revealed no histological abnormalities in skeletal muscle until ~6 mo of age. However, 1-yr-old Tg2 mice showed a few faintly stained degenerative myofibers in various muscles. The number of centronucleated myofibers in 1-yr-old Tg2 mice was also significantly increased compared with wild-type mice of the same age (2.5 ± 0.5 vs. 0.2 ± 0.1%).


Figure 4
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  		Fig. 4. Progressive myofiber degeneration of Tg1 mice. Hematoxylin and eosin staining of gastrocnemius muscles of Tg1 mice at the age of 20 days, 1 mo, and 6 mo. At the age of 1 mo, faintly stained degenerative myofibers (arrow) and centronucleated myofibers appeared in the Tg1 mouse, in contrast to the WT littermate. Arrowheads indicate the infiltration of mononuclear cells. At the age of 6 mo, centronucleated myofibers were more predominant, with prominent differences in size. Fiber splitting (arrowhead) is also indicated. No histological abnormalities were noted at the age of 20 days. Scale bars: 40 µm.

 
We measured serum CK activity in 3-mo-old Tg-1 mice and their wild-type littermates because the measurement of serum CK activity is used clinically to ascertain muscular damage. Tg1 mice showed significantly higher serum CK activity than the wild-type littermates (P < 0.01) (Fig. 5A). Evans blue dye labeling also detected more clearly the damaged myofibers, with increased membrane permeability in Tg1 mice and aged 1-yr-old Tg2 mice (Fig. 5B).


Figure 5
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  		Fig. 5. Evaluation of membrane permeability of Tg1 skeletal muscle. A: serum creatine kinase (CK) activity of Tg1 mice at the age of 3 mo. Tg1 mice showed significantly higher CK activity than WT littermates (P < 0.01). B: after Evans blue dye injections, some myofibers of Tg1 mice and 1-yr-old Tg2 mice accumulated the dye in cytoplasm, showing increased membrane permeability. Scale bars: 40 µm.

 
Evaluation of causative proteins of muscular dystrophy. These skeletal muscle phenotypes caused by mBD-6 overexpression are reminiscent of muscular dystrophies, characterized by progressive myofiber degeneration. In mBD-6 transgenic mice, organization of the dystrophin-glycoprotein complex was not different from that of wild-type littermates on the immunohistochemistry of dystrophin, {alpha}-dystroglycan, and laminin (Fig. 6). Likewise, the expression of calpain-3 showed no abnormality in mBD-6 transgenic mice (Fig. 6).


Figure 6
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  		Fig. 6. Immunohistochemical analyses of dystrophin, {alpha}-dystroglycan, laminin, and calpain-3 distributions in Tg1 skeletal muscle. Distribution of these molecules in Tg1 mice showed no difference from WT. Dystrophin is absent in dystrophin-deficient muscle (mdx). Scale bars: 20 µm.

 
Immunohistochemical abnormalities of the transgenic myofibers. To investigate the molecular mechanisms of myofiber degeneration, we evaluated the conserved immunohistochemical features of young Tg1 mice and aged Tg2 mice.

In 1-mo-old Tg1 mice, many myofibers showed a high level of expression of neural cell adhesion molecule (NCAM). Also, in Tg2 mice, many NCAM-positive myofibers were detected at 11–12 mo of age, while their wild-type littermates of the same age showed only a few NCAM-positive myofibers (Fig. 7A). Although denervated myofibers upregulate NCAM expression, the number and morphology of motor neurons were not different between the Tg1 mice and their wild-type littermates (Fig. 8). We also examined the distribution of I{kappa}B{alpha} in Tg1 and Tg2 mice. We detected the accumulation of I{kappa}B{alpha} in many myofibers of 1-mo-old Tg1 mice and 12-mo-old Tg2 mice (Fig. 7B), as reported in LGMD2A patients (2). We also evaluated the apoptotic features of I{kappa}B{alpha}-positive myofibers in transgenic mice. In the staining of serial sections, some of the I{kappa}B{alpha}-positive myofibers showed the signal of cleaved caspase-3, the active form of caspase-3, indicating activation of the apoptotic pathway (Fig. 9).


Figure 7
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  		Fig. 7. Immunohistochemical analyses of neural cell adhesion molecule (NCAM) and I{kappa}B{alpha} distributions in young Tg1 mice and aged Tg2 mice. A: many myofibers showed a high level of expression of NCAM in 1-mo-old Tg1 mice and 12-mo-old Tg2 mice, in contrast to WT littermates. B: many myofibers showed accumulation of I{kappa}B{alpha} in 1-mo-old Tg1 mice and 12-mo-old Tg2 mice, in contrast to WT littermates. Scale bars: 40 µm.

 

Figure 8
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  		Fig. 8. Hematoxylin and eosin staining of spinal cords of Tg1 mouse and WT littermate. No. and morphology of motor neurons showed no abnormality causative of myofiber degeneration in the Tg1 mouse.

 

Figure 9
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  		Fig. 9. Apoptotic feature of Tg2 skeletal muscle. Immunohistochemical analysis of serial sections for I{kappa}B{alpha} and cleaved caspase-3 indicated that some myofibers (arrow) showed both accumulation of I{kappa}B{alpha} and apoptotic features. Scale bars: 40 µm.

 
Other pathological changes and food intake in the mBD-6 overexpressing mouse. mBD-6 overexpression decreases body weights and increases centronucleated myofibers and degenerative myofibers in mice. Kyphosis of vertebra, shortness in height, and slow movement were observed in the mouse. The food intake of Tg1 mice was significantly lower than that of the wild-type littermates. The short lifespan of Tg1 mice was evident, especially after they were backcrossed to the C57BL6/J strain. Many Tg1 mice died before 8 mo, and no mice backcrossed to the C57BL6/J strain lived for >1 yr. There was no evidence of cancer in the dead Tg1 mice.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data first demonstrated the pathogenic effects of dysregulated beta-defensin expression in vivo. Western blot analysis of muscle extracts ascertained the overproduction of mature mBD-6 peptide in the Tg1 and Tg2 mice. The dysregulated beta-defensin expression induced poor growth, short lifespan, and functional muscle impairment. Pathologically, the skeletal muscle of Tg1 mice showed progressive degeneration and regeneration of myofibers, consistent with the histology of muscular dystrophy. Elevated serum CK activity and positive Evans blue dye labeling in Tg1 mice indicated the disruption of the myofiber plasma membrane, also consistent with muscular dystrophy.

Despite recent identification of causative genes, the clinical course of muscular dystrophy is miserable lacking an established therapy. Although gene therapy could be curative, replacing the ultimate defect, many obstacles exist to technical progress. So, presently, important therapy targets are the factors modulating the state of muscle degeneration. Clinically, glucocorticoids are utilized to delay the progression of Duchene muscular dystrophy (12, 20, 34), and, actually, the invasion of lymphoid and myeloid cells is an early stage feature of Duchene muscular dystrophy (33). beta-Defensin would be the first reported component of inflammation that induced alone the typical phenotype of muscular dystrophy. Because mBD-6 and human defensin-3 showed intrinsic expression in skeletal muscle (10, 39), and invaded myeloid cells and lymphocytes would secrete abundant {alpha}-defensin in human muscular dystrophy (42), our findings suggest the significance of the defensin family in the pathogenesis of muscular dystrophy.

In aged Tg2 mice, mBD-6 overexpression induced NCAM-positive myofibers and I{kappa}B{alpha} accumulation with mild histological abnormality. Interestingly, aging alone induced a slight increase in NCAM-positive myofibers and I{kappa}B{alpha}-positive myofibers. The augmentation of these aging phenomena in Tg2 mice suggested that defensin-mediated muscle degeneration would not be limited to distinct muscular dystrophy but would be associated with a much more common late-onset muscular wasting degeneration like sarcopenia, cachexia, or senescence acceleration.

Previous studies with transgenic mice overexpressing antimicrobial proteins (e.g., lysozyme) or peptides (e.g., human defensin-5) have indicated the beneficial roles of these substances in the in vivo situation (1, 27). In contrast to these animal models, Tg1 mice succumbed to muscular degeneration and short lifespan, showing a completely novel aspect of antimicrobial peptides. The various mediators, like reactive oxygen metabolites, complement cascades, and some proteases are involved in the known immune-mediated tissue injury in inflammatory conditions. Our investigation has established the defensin family as a novel effector of immune-mediated tissue injury.

The molecular mechanism of defensin-mediated tissue injury remains to be clarified. Generally, pore formation and permeabilization of target membranes are common mechanisms of defensin effects (9, 18, 19). Because dystrophin, dystroglycan, and laminin distributions were normal, mBD-6 would induce muscle degeneration independently of the dystrophin-glycoprotein complex.

Although upregulation of NCAM was shown in denervated myofibers, the histology of motor neurons of Tg1 mice showed no abnormality that could be causative for massive myofiber degeneration. So, these NCAM-positive myofibers would indicate the regenerative process and/or association of motor endplate degeneration in Tg1 and Tg2 mice, as indicated in some types of muscular dystrophy (7, 13, 38). At the same time, the accumulation of I{kappa}B{alpha} in Tg1 and Tg2 mice with apoptotic features is similar to that in LGMD2A patients and their animal models (2, 25). In LGMD2A patients, the defect of I{kappa}B{alpha} turnover inhibited the activity of NF-{kappa}B, associated with myofiber apoptosis. Because perturbation of the NF-{kappa}B/I{kappa}B pathway could cause the subsequent perturbation of many survival genes, it could be a common pathway in various mechanisms of myofiber degeneration.

The next question is, what downstream events of the beta-defensin-6 pathway might be involved in the mechanisms of myofiber degeneration? Actually, both CCR6 and TLR4 are expressed in skeletal muscle. Other investigators (8) have reported that TLR4 expression is identified in skeletal muscles. We also confirmed the expression of TLR4 using RT-PCR. However, we do not think that the downstream events of the beta-defensin-6 pathway are important in the pathogenesis of the muscle degeneration phenotype. Until now, there have been no data about the pathological relationship between muscle degeneration and TLRs. While various functions of the defensin family in mammalian cells have been reported, including cytotoxicity, most of the downstream events were not clarified, and the contribution of CCR6 or TLR4 on the pathogenesis of muscle degeneration would be limited, if it existed. Certainly, CCR6 receptors and TLR4 are expressed in skeletal muscle, and the NF-{kappa}B pathway is directly associated with TLR4. However, it is reasonable to speculate that continuous stimulation of these receptors does not primarily contribute to induce degeneration of myofibers in vivo. If such a phenomenon were to happen, the muscles could be totally abolished, and severe muscle damage would occur earlier in life.

In this study, body weights of Tg1 mice were significantly lower than for wild-type mice. Food intake of Tg1 mice was significantly lower than for wild-type littermates. The decreased food intake could explain, in part, decreased body weight. However, prominent myofiber degeneration could never be induced by decreased food intake, and it is more likely that decreased food intake resulted from decreased muscular mass and strength. Furthermore, Tg1 mice had a short life span. Most Tg1 mice died before 11 mo of age. The short lifespan of Tg1 mice was especially evident after they were backcrossed to the C57Bl6/J strain. Many Tg1 mice died before 8 mo, and no mice backcrossed to the C57BL6/J strain lived for >1 yr. We could not clarify the specific cause of Tg1 mouse death except severe loss of body weight. There was no evidence of cancer in the dead Tg1 mice; thus we speculate that impaired immune function may cause systemic inflammation and decreased food intake at a relatively young age, resulting in a malnutrition-related short lifespan.

In conclusion, our study has demonstrated that the defensin family could contribute to the pathogenic immune response in animal models, especially in the pathogenesis of myofiber degeneration.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, Grants-in-Aid for Comprehensive Research on Aging and Health from the Ministry of Health, Labor and Welfare of Japan, and a Research Grant from Uehara Memorial Foundation.


   ACKNOWLEDGMENTS
 
We thank H. Sugita and I. Nonaka for helpful discussion. We also thank H. Sorimachi for providing calpain-3 antibody and J. Miyazaki for the pCAGGS plasmid.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Ouchi, Dept. of Geriatric Medicine, Graduate School of Medicine, The Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan (e-mail: youchi-tky{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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I. Nagaoka, F. Niyonsaba, Y. Tsutsumi-Ishii, H. Tamura, and M. Hirata
Evaluation of the effect of human {beta}-defensins on neutrophil apoptosis
Int. Immunol., April 1, 2008; 20(4): 543 - 553.
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