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Distinct roles for Ste20-like kinase SLK in muscle function and regeneration.

Storbeck CJ, Al-Zahrani KN, Sriram R, Kawesa S, O'Reilly P, Daniel K, McKay M, Kothary R, Tsilfidis C, Sabourin LA - Skelet Muscle (2013)

Bottom Line: 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.Together, these results suggest complex and distinct roles for SLK in muscle development and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Hospital Research Institute, 501 Smyth Rd, Box 926, Ottawa, ON K1H8L6, Canada. lsabourin@ohri.ca.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Generation of transgenic lines. (A) Schematic representation of the hemagglutinin-tagged Ste20-like kinase (SLK) construct. Full-length murine SLK mutated at the ATP-binding site (K63R) is driven by 2500 bp of upstream promoter sequences derived from the human skeletal actin gene. A SV40 polyadenylation signal was also cloned downstream of SLK (not shown). The 1.9 kb PvuII probe fragment spanning the promoter and cDNA regions is shown. (B) Representative Southern blot analysis of a full litter from line 3405 crossed with wild-type FVB/N showing the transgene signal on a PvuII digest of tail DNA. The corresponding PCR analysis is shown below the autoradiogram.
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Figure 1: Generation of transgenic lines. (A) Schematic representation of the hemagglutinin-tagged Ste20-like kinase (SLK) construct. Full-length murine SLK mutated at the ATP-binding site (K63R) is driven by 2500 bp of upstream promoter sequences derived from the human skeletal actin gene. A SV40 polyadenylation signal was also cloned downstream of SLK (not shown). The 1.9 kb PvuII probe fragment spanning the promoter and cDNA regions is shown. (B) Representative Southern blot analysis of a full litter from line 3405 crossed with wild-type FVB/N showing the transgene signal on a PvuII digest of tail DNA. The corresponding PCR analysis is shown below the autoradiogram.

Mentions: We have previously shown that SLK is highly expressed in both the neuronal and myogenic compartment in the developing embryo [31]. In addition, expression of a kinase-inactive SLK in C2C12 cells inhibits myoblast fusion [32]. Together, these data suggest a role for SLK in muscle differentiation and function. To gain further insight into the role of SLK in differentiated muscles, we generated a skeletal actin-driven transgene (Figure 1). The HA-tagged kinase-inactive SLK (K63R) transgene was purified and injected into donor zygotes. Using Southern blot analysis and a transgene-specific probe (Figure 1), 5 founders were identified from 45 mice surveyed. The presence of the transgene was further confirmed by PCR analysis (Figure 1). Two transgenic lines were then derived from independent founders for further analysis.


Distinct roles for Ste20-like kinase SLK in muscle function and regeneration.

Storbeck CJ, Al-Zahrani KN, Sriram R, Kawesa S, O'Reilly P, Daniel K, McKay M, Kothary R, Tsilfidis C, Sabourin LA - Skelet Muscle (2013)

Generation of transgenic lines. (A) Schematic representation of the hemagglutinin-tagged Ste20-like kinase (SLK) construct. Full-length murine SLK mutated at the ATP-binding site (K63R) is driven by 2500 bp of upstream promoter sequences derived from the human skeletal actin gene. A SV40 polyadenylation signal was also cloned downstream of SLK (not shown). The 1.9 kb PvuII probe fragment spanning the promoter and cDNA regions is shown. (B) Representative Southern blot analysis of a full litter from line 3405 crossed with wild-type FVB/N showing the transgene signal on a PvuII digest of tail DNA. The corresponding PCR analysis is shown below the autoradiogram.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733878&req=5

Figure 1: Generation of transgenic lines. (A) Schematic representation of the hemagglutinin-tagged Ste20-like kinase (SLK) construct. Full-length murine SLK mutated at the ATP-binding site (K63R) is driven by 2500 bp of upstream promoter sequences derived from the human skeletal actin gene. A SV40 polyadenylation signal was also cloned downstream of SLK (not shown). The 1.9 kb PvuII probe fragment spanning the promoter and cDNA regions is shown. (B) Representative Southern blot analysis of a full litter from line 3405 crossed with wild-type FVB/N showing the transgene signal on a PvuII digest of tail DNA. The corresponding PCR analysis is shown below the autoradiogram.
Mentions: We have previously shown that SLK is highly expressed in both the neuronal and myogenic compartment in the developing embryo [31]. In addition, expression of a kinase-inactive SLK in C2C12 cells inhibits myoblast fusion [32]. Together, these data suggest a role for SLK in muscle differentiation and function. To gain further insight into the role of SLK in differentiated muscles, we generated a skeletal actin-driven transgene (Figure 1). The HA-tagged kinase-inactive SLK (K63R) transgene was purified and injected into donor zygotes. Using Southern blot analysis and a transgene-specific probe (Figure 1), 5 founders were identified from 45 mice surveyed. The presence of the transgene was further confirmed by PCR analysis (Figure 1). Two transgenic lines were then derived from independent founders for further analysis.

Bottom Line: 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.Together, these results suggest complex and distinct roles for SLK in muscle development and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ottawa Hospital Research Institute, 501 Smyth Rd, Box 926, Ottawa, ON K1H8L6, Canada. lsabourin@ohri.ca.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.