Distinct roles for Ste20-like kinase SLK in muscle function and regeneration
© Storbeck et al.; licensee BioMed Central Ltd. 2013
Received: 24 October 2012
Accepted: 2 May 2013
Published: 1 July 2013
Cell growth and terminal differentiation are controlled by complex signaling systems that regulate the tissue-specific expression of genes controlling cell fate and morphogenesis. We have previously reported that the Ste20-like kinase SLK is expressed in muscle tissue and is required for cell motility. However, the specific function of SLK in muscle tissue is still poorly understood.
To gain further insights into the role of SLK in differentiated muscles, we expressed a kinase-inactive SLK from the human skeletal muscle actin promoter. Transgenic muscles were surveyed for potential defects. Standard histological procedures and cardiotoxin-induced regeneration assays we used to investigate the role of SLK in myogenesis and muscle repair.
High levels of kinase-inactive SLK in muscle tissue produced an overall decrease in SLK activity in muscle tissue, resulting in altered muscle organization, reduced litter sizes, and reduced breeding capacity. The transgenic mice did not show any differences in fiber-type distribution but displayed enhanced regeneration capacity in vivo and more robust differentiation in vitro.
Our results show that SLK activity is required for optimal muscle development in the embryo and muscle physiology in the adult. However, reduced kinase activity during muscle repair enhances regeneration and differentiation. Together, these results suggest complex and distinct roles for SLK in muscle development and function.
KeywordsSte20-like Kinase Muscle Regeneration Transgenic
Growth and differentiation of muscle cells are regulated by complex processes involving a large number of signaling systems. Activation or inhibition of various pathways results in the expression of specific subsets of genes directly involved in proliferation or terminal differentiation [1–5]. In yeast, the serine/threonine protein kinase Ste20 regulates a mitogen- activated protein kinase pathway consisting of the Ste11 protein kinase (a mitogen-activated protein kinase kinase; MEKK), Ste7 protein kinase (a mitogen-activated protein kinase kinase; MEK), and Fus3/Kss1 protein kinase (a mitogen-activated protein kinase; MAPK) involved in the control of mating response . Ste20 has also been shown to bind the small GTPase Cdc42, but its Cdc42-binding domain has been shown to be dispensable for pheromone signaling in yeast . Several members of the Ste20 family of kinases have been identified in mammals , and have been shown to play a role in various biological processes such as stress, cell death, cytoskeletal reorganization, growth, and differentiation [9–15]. A novel Ste20-related kinase was previously identified  and termed Ste20-like serine/threonine protein kinase (SLK) [17–19]. Overexpression of SLK has been shown to induce breakdown of actin stress fibers and cell death in various systems [19–22]. A role for SLK in cell migration and cell-cycle progression has also been shown [23–30].
During murine embryogenesis, SLK is preferentially expressed in muscle and neuronal lineages . Despite a role for SLK in cell death, it is also expressed at high levels in muscle tissues and proliferating myoblasts, suggesting a functional role for this kinase in physiological processes other than apoptosis . Our previous data showed that SLK is expressed in the muscle mass of developing embryos and is found at myofibrillar striations of specific subsets of myofibers [31, 32]. Furthermore, expression of dominant negative SLK in C2C12 myoblasts inhibits terminal differentiation . To gain further insights into the role of SLK in myogenic development, we characterized transgenic animals expressing a kinase-inactive SLK mutant from the human skeletal actin promoter. Our results showed that muscle-specific expression of a dominant negative SLK reduces overall kinase activity in muscle tissue, and affects muscle development and litter size. Interestingly, transgenic animals showed enhanced regenerative capacity in vivo and increased differentiation potential in vitro. These results suggest complex and distinct roles for SLK in differentiation and function of muscle cells.
Animal studies were approved by the University of Ottawa animal ethics board. Care and use of experimental mice followed the guidelines established by the Canadian Council on Animal Care.
Transgenic plasmid DNA was constructed by inserting the human skeletal actin promoter (-2500 bp)  upstream of full-length (3600 bp) kinase-inactive SLK bearing a point mutation at lysine 63 (K63R) . This ATP-binding site mutation inactivates kinase activity in an autophosphorylation assay . Injection and derivation of transgenic mice were performed using linearized plasmid DNA as previously described .
Primers for PCR amplification
Tissue collection and analysis
For muscle injury, mice aged 8 to 10 weeks old were anesthetized, and cardiotoxin 10 μmol/l was injected into the belly of the tibialis anterior (TA) muscle . The mice were allowed to recover and the muscles were collected at 7 days post-injection. The tissues were embedded in optimal cutting temperature compound, and cryosectioned for hematoxylin and eosin (H&E) staining . To assess muscle damage, the cross-sectional area (CSA) of the regenerating fibers was measured from random fields (ImageScope; Aperio, Vista, CA, USA). Data are presented as the proportion of fibers within a specific range of CSA for both transgenic lines. Embryos were collected by caesarean section of timed matings and genotyped using placental DNA. For immunostaining, embryos and TA muscles were removed and fixed in 4% paraformaldehyde (PFA), followed by perfusion in 10% sucrose. The tissues were then frozen in isopentane, cut into12 μm sections, and assayed by immunochemistry. Embryos or muscle sections were stained with MyoD (sc304; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Myogenin (F5D) and Pax7 (1E12). The Myogenin and Pax7 monoclonals were used as hybridoma supernatants (Developmental Studies Hybridoma Bank, Iowa City, IA). Fiber-type-specific monoclonal antibodies consisted of Hybridoma Bank clones SC-71 (type IIA), BF-F3 (type IIB), and A4-840 (type I) (all kind gift of Dr Robert Parry, University of Ottawa).
For western blotting and kinase assays, lower anterior muscles or cardiac tissue were removed and ground in liquid nitrogen. The tissue powder was then lysed in RIPA buffer as previously described , and lysates were cleared by centrifugation at 10,000 g for 2 minutes. Protein concentrations were determined using protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (20 to 40 μg) were separated by electrophoresis on 8 to 15% polyacrylamide gels, and transferred to PVDF membranes. Membranes were probed with anti-hemagglutinin (HA; 12CA5) or anti-SLK antibodies overnight at 4°C in 5% skim milk powder in 1 × Tris-buffered saline with Tween (TBS-T; 50 mmol/l Tris pH 7.4, 150 mmol/l NaCl, and 0.05 Tween 20). Membranes were washed in TBS-T and the reactive proteins were detected using chemiluminescence (Perkin Elmer, Waltham, MA, USA) and exposure to X-ray film.
For immunoprecipitations, 300 μg of protein lysate was immunoprecipitated with 2 μg of antibody and 20 μl of protein A sepharose (Pharmacia & Upjohn Inc., Bridgewater, NJ, USA) for 2 to 12 hours. Immune complexes were recovered by centrifugation and washed with NETN buffer (20 mmol/l Tris–HCl pH 8.0, 1 mmol/l EDTA, 150 mmol/l NaCl, 0.5% Nonidet P-40), then used for SDS-polyacrylamide gel electrophoresis (PAGE) or kinase assays. In vitro kinase assays were performed following SLK immunoprecipitation as described previously , transferred to PVDF membranes and used for autoradiography, followed by western blotting with SLK antibody .
Satellite-cell cultures and in vitro differentiation
Only myotubes containing three or more nuclei were scored.
For western blot analyses cultures were lysed as above and probed with MF20, MyoD, myogenin and cyclin D1 (sc20044; Santa Cruz Biotechnology) antibodies.
Generation of SLK transgenic mice
To further investigate the embryonic phenotype, transgenic and wild-type embryos from timed matings were collected for analysis. The embryos (11.5 and 13.5 days post-conception (dpc)) were cryosectioned and used for MF20 or myogenin immunostaining. MF20-positive fibers were present in both wild-type and transgenic 13.5 dpc embryos (Figure 3B-E). However, muscle fibers appeared to be significantly larger in the wild-type animals. The wild-type muscles displayed thick parallel bundles of myofibers, whereas the high-expressing transgenic mice (line 654) had a more disorganized musculature (Figure 3B-E). Immunohistochemical analysis for myogenin, an early myogenic marker [41, 42], showed relatively smaller pre-muscle masses in the high-expressing 654 line at 11.5 dpc (Figure 3F,G). As for older embryos, MF20 analysis of 11.5 dpc embryos showed smaller myofibers and reduced pre-muscle masses (Figure 3H,I).
These results suggest that muscle development may be delayed in high-expressing embryos. As it displayed a more robust phenotype, only the 654 line was further characterized.
Altered regeneration in transgenic muscles
To further investigate the mechanism responsible for enhanced differentiation in vitro, myoblast cultures from transgenic and wild-type animals were differentiated and assessed for the expression of differentiation markers. Supporting the in vitro fusion data, MHC levels were markedly increased in HA-K63R cultures at day 2 after onset of differentiation (four-fold versus >100-fold; Figure 6D). However, myogenin and MyoD levels did not show any appreciable differences. Surprisingly, transgenic cultures showed a marked downregulation of cyclinD1 levels as they proceeded through differentiation. Wild-type cultures had a two-fold reduction in cyclin D1 over the time course, whereas a ten-fold downregulation was seen in the transgenic cultures (Figure 6D). Together, these data suggest that the HA-K63R myoblasts have enhanced differentiation potential in vitro. and that they can exit the cell cycle much more efficiently.
Using transgenic lines expressing a kinase-inactive SLK from the human skeletal actin promoter, we have shown that high levels of dominant-negative SLK result in impaired development and accelerated differentiation in vivo following muscle injury. Similarly, myoblast cultures derived from transgenic mice differentiate more efficiently in vitro. These data suggest potentially complex and distinct roles for SLK in embryonic and adult muscles.
Muscle-cell differentiation and myoblast fusion is regulated by complex signaling networks [3, 43]. Myoblast fusion is also highly dependent on cytoskeletal remodeling and on factors controlling actin dynamics and adhesion [44–53]. We have recently shown that the Ste20-like kinase SLK is required for efficient cell migration, chemotaxis, and focal adhesion turnover [24, 26, 27, 54]. Our previous findings showed that expression of kinase-inactive SLK in myoblasts impaired fusion . To further investigate its role in skeletal muscle in the current study, we generated transgenic mice expressing kinase-inactive SLK from the human skeletal actin promoter. Immunoprecipitation and western blotting analysis showed that in transgenic animals the overall levels of SLK were increased two-fold to four-fold. However, the overall kinase activity was markedly reduced, suggesting that HA-K63R has a dominant-negative effect. SLK has recently been reported to function as a homodimer [37, 55]. Furthermore, autophosphorylation of the activation loop seems to be required for maximal kinase activity. Therefore, it is likely that the HA-K63R version can associate with endogenous SLK, preventing full activation as a result of lack of complete autophosphorylation. This dominant-negative phenotype is therefore likely to be contributing to the delayed development of the higher-expressing 654 line.
Interestingly, mice that are deficient for both MyoD and Myf5 develop until birth [56, 57]. As the transgene is not detected in cardiac tissues (Figure 2), it is unclear how muscle-specific K63R transgene expression induces embryonic lethality. It is possible that high expression of kinase-inactive SLK in pre-muscle masses is detrimental. However, a more likely explanation is that the 654 line has undergone a chromosomal rearrangement of a crucial gene, responsible for this apparent dominant embryonic lethality. These anomalies have been shown to result in reduced litter sizes and arrested development before 7 dpc .
Our previous data showed that SLK is preferentially expressed in type I myofibers . Interestingly, HA-K63R-expressing mice displayed a reduced proportion of large type I fibers, suggesting a possible role for SLK in the maintenance of these fibers. Several adhesion proteins such as focal adhesion kinase (FAK) and paxillin have been implicated in muscle organization and function [58–60]. As SLK is activated downstream of FAK-mediated motility signaling [24, 27], one possibility is that expression of HA-K63R suppresses further signals, leading to maturation defects and atrophy.
Using C2C12 cells, we previously found that expression of a truncated kinase-inactive SLK in myoblasts inhibits fusion in a cell autonomous manner . Surprisingly, expression of dominant-negative SLK from the skeletal actin promoter enhanced muscle regeneration after cardiotoxin-induced damage. Similarly, myoblast cultures derived from HA-K63R-expressing mice displayed increased differentiation potential, as evidenced by higher fusion indices and increased levels of MHC protein. As SLK is required both for proliferation  and cytoskeletal dynamics , these observations raise the possibility that SLK plays different roles during myoblast differentiation. Supporting this, SLK kinase activity is downregulated upon serum withdrawal from C2C12 cultures, but upregulated in differentiated myotubes . As myoblast proliferation and differentiation are mutually exclusive , one possibility is that HA-K63R expression in differentiating myocytes facilitates cell-cycle exit, enhancing differentiation. Supporting this hypothesis is the observation that differentiating transgenic cultures show marked downregulation of cyclin D1 levels, suggesting that they exit the cell cycle much more efficiently than do wild-type cells. Surprisingly, fusion is enhanced in HA-K63R-derived myoblasts. It is possible that the residual low level of kinase activity is sufficient to allow fusion to proceed.
Together with our previous results , these data suggest a complex mechanism by which SLK is required for cytoskeletal dynamics before fusion, then is downregulated for cell-cycle exit but re-activated for muscle-specific functions. Identification of SLK substrates and generation of SLK knockout models will further help to delineate between these possibilities.
Dulbecco’s modified Eagle’s medium
Focal adhesion kinase
Haematoxylin and eosin
Mitogen-activated protein kinase
Myosin heavy chain
Polyacrylamide gel electrophoresis
Phosphate buffered saline
Sodium dodecyl sulfate
Tris-buffered saline with Tween.
This work was supported by the Canadian Institute for Health Research and MDAUSA. CJS is the recipient of a Canadian Heart and Stroke Foundation Fellowship. KAZ is funded by the Canadian Breast Cancer Foundation. PO is the recipient of an OGSST studentship. The authors declare no conflict of interest.
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