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REPORT
MUSCLE CELL BIOLOGY AND CELL MOTILITY
-catenin is necessary for physiological growth of adult skeletal muscle
Department of Physiology, University of Kentucky, Lexington, Kentucky
Submitted 22 December 2005 ; accepted in final form 23 January 2006
ABSTRACT
Expression of 
-catenin is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Inappropriate expression, however, has been linked to pathological states such as cancer. Here we report that expression of -catenin is necessary for physiological growth of skeletal muscle in response to mechanical overload. Conditional inactivation of -catenin was induced in control and overloaded muscle through intramuscular injection of adenovirus expressing Cre recombinase in -catenin floxed mice. Individual muscle fiber analysis was performed to identify positively transfected/inactivated cells and determine fiber cross-sectional area. The results demonstrate that fiber growth is completely inhibited when the -catenin expression is lost. This effect was cell autonomous, as fibers that did not exhibit recombination in the floxed mice grew to the same magnitude as infected/noninfected fibers from wild-type mice. These findings suggest that -catenin may be a primary molecular site through which multiple signaling pathways converge in regulating physiological growth.
hypertrophy; Wnt; overload
Although these pathways are normally studied in isolation, it has become apparent that there are both synergistic and convergent interactions among pathways (15). One site of convergence in models of growth is with the expression of -catenin through inhibition of glycogen synthase kinase-3 (GSK-3) (4, 7, 12). Subsequent to GSK-3 inhibition, -catenin accumulates in the cytoplasm, where it can interact with cadherin at the membrane or it can be translocated to the nucleus (12, 15). In the nucleus, -catenin interacts with transcriptional coactivators, such as members of the T cell factor/lymphocyte enhancement factor-1 (Tcf/Lef-1) family and induces expression of growth-associated genes such as c-myc and cyclin D1 (13, 14). Overexpression of -catenin in myocytes and other nonmuscle cells has been shown to be sufficient to support cellular growth (9, 11, 13); however, it is not clear whether -catenin is a necessary molecular target for regulating growth in vivo.
This study used mechanical overload, a well-characterized physiological model, to induce in vivo growth in adult skeletal muscle. In this model, two of three synergistic muscles of the posterior hindlimb are removed, which induces a robust and reliable fiber growth in response to the increased loading in the remaining muscle (1, 2, 5, 8). Because -catenin is necessary for skeletal muscle development (6), inducible gene deletion was used to examine the role of -catenin in adult skeletal muscle growth. Conditional recombination of the -catenin gene was accomplished by injecting control (Cnt) and overloaded (Ovl) adult skeletal muscles of wild-type (WT) and floxed -catenin (BC) mice (Jackson Laboratory, Bar Harbor, ME) with an adenovirus expressing both Cre recombinase and green fluorescent protein (GFP) (Adv-Cre-GFP). We obtained an E1/E3-deleted, replication-incompetent, serotype 5 adenovirus-expressing Cre recombinase and GFP under control of the cytomegalovirus (CMV) promoter from the Baylor Vector Development Laboratory. All experimental procedures performed in this study were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Adenovirus-mediated gene delivery has a low and variable rate of infection when used in mature skeletal muscle (10). The first set of experiments determined the efficacy of infection and recombination on inactivation of -catenin in skeletal muscle. Cnt and Ovl plantaris muscles were perfusion fixed (20 ml PBS and 4% Formalin) in vivo at 14 days after injection, sectioned, and immunohistochemically probed for GFP expression. As listed in Table 1 and seen in Fig. 1A, 
18% of examined muscle fibers in all groups were found to be GFP positive (GFP+) after Adv-Cre-GFP injection. Adenoviral infection is also known to induce an immune response, so hematoxylin and eosin-stained sections were compared to determine whether there was an increased immune response (Fig. 1B). As expected, all muscles injected with Adv-Cre-GFP exhibited regions of increased mononuclear cell infiltration, but there was no qualitative difference in the magnitude of the response when comparing injected Cnt vs. Ovl muscles at 14 days.
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-catenin transgene. An appropriate-size floxdel product (631 bp) was detected in BC mice (Fig. 2A). Individual fiber analysis was performed immunohistochemically to determine whether -catenin gene recombination was associated with fiber infection. As shown in Fig. 2B, GFP+ muscle fibers were identified from injected BC Ovl muscle sections and these fibers stained negative for -catenin expression. This provides direct evidence that infected muscle fibers in BC mice did exhibit loss of -catenin expression. Collectively these experiments confirm that adenoviral injection of Cre recombinase mediated gene recombination of the -catenin locus, and loss of -catenin expression was detectable at the single-fiber level.
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-catenin in response to mechanical overload, single-muscle fiber analysis was done for cross-sectional area (CSA) of both infected/GFP+ and noninfected/GFP– fibers. Two x20 magnification images were captured from five individual muscles and analyzed for CSA with SPOT software by an investigator blinded to the genotype and muscle treatment group. Over 4,000 fibers were analyzed per group, and the results were statistically analyzed with a three-way analysis of variance (Table 1). The mosaic nature of the adenoviral infection in adult skeletal muscle means that infected fibers are located next to uninfected fibers in the same muscle. The power of this type of design is that quantitative comparisons can be made within muscles of a particular genotype and treatment group so that fibers that are exposed to the exact same physiological environment and only vary based on the presence or absence of inactivation can be compared.
The results from our analysis demonstrate that -catenin is necessary for in vivo growth of differentiated adult skeletal muscle fibers. The evidence to support this conclusion comes from quantitative comparisons of data in Table 1 and is illustrated in Fig. 3. We found that, unlike the 60% increase in CSA seen in Ovl WT mice (P < 0.05), there is no difference in fiber CSA between Cnt and Ovl GFP+ fibers in BC mice.
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-catenin on the growth response is fiber autonomous, as the neighboring nontransfected/GFP– fibers in Ovl BC mice were not different in size from GFP+/GFP– fibers in Ovl WT mice. In addition, the GFP– fibers in Ovl BC mice were significantly larger than all fibers in Cnt BC mice. We were surprised that there was no detectable growth in the recombined/GFP+ fibers of Ovl BC mice. It has been well established that the IGF-I/PI3-kinase/Akt pathway is activated in this model of mechanical overload, and this pathway would still be intact in the -catenin knockout/GFP+ fibers (1, 3, 5, 16). Thus our findings suggest that either 1) enhanced expression of -catenin is a common molecular target at which multiple signaling pathways converge in directing skeletal muscle growth or 2) -catenin can act upstream of PKB signaling. Although we cannot rule out option 2 with the data presented, to date there is no evidence suggesting that -catenin acts upstream to regulate PKB signaling.
In summary, the results of this study demonstrate that expression of -catenin is absolutely necessary for in vivo skeletal muscle fiber growth. In addition, the fiber-autonomous effect of loss of -catenin expression in response to overload suggests that -catenin may be a fundamental molecular site through which multiple signaling pathways converge and synergistically regulate muscle fiber growth. Depending on whether the goal is to promote cell growth, as in skeletal muscle of the aging, or to inhibit growth, in the case of cancer, these findings implicate the manipulation of -catenin levels as a likely target for therapeutic design.
GRANTS
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45617 (to K. A. Esser).
ACKNOWLEDGMENTS
We thank Dr. A. Stromberg for assistance with the statistical analysis and Dr. J. McCarthy for helpful discussions.
Present address for D. D. Armstrong: Novartis Institutes for BioMedical Research, Inc., Models of Disease Center, Epigenetics Dept., 250 Massachusetts Ave., 4C-341, Cambridge, MA 02139 (e-mail: dustin.armstrong{at}novartis.com).
FOOTNOTES
Address for reprint requests and other correspondence: K. A. Esser, Dept. of Physiology, MS567 Medical Center, 800 Rose St., Lexington, KY 40536-0298 (e-mail: karyn.esser{at}uky.edu)
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.
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P < 0.0001) and BC Ovl– fibers within the same muscle section (819 ± 127 vs. 1,390 ± 241 µm2; 
P < 0.0001). Furthermore, GFP+ fibers from BC Ovl (B) are not significantly different from GFP+ fibers of BC Cnt muscle (A). Values represent means ± SE (see numbers in