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NF-kappaB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells.

Wang H, Yang S, Yang E, Zhu Z, Mu Y, Feng S, Li K - BMC Mol. Biol. (2007)

Bottom Line: In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1.These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells.The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gene and Cell Engineering, State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, PR China. muwhy@hotmail.com <muwhy@hotmail.com>

ABSTRACT

Background: The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1) is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2) and calsarcin-3 (CS-3) is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated.

Results: Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-kappaB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-kappaB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1.

Conclusion: Our present data suggest that NF-kappaB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.

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Reporter analysis of the mouse CS1 and CS2 gene promoters. A. Reporter analysis of mouse CS1 gene promoter deletion constructs. B. Reporter analysis of mouse CS2 gene promoter deletion constructs. The 5'-deleted promoter segments were generated as described in Materials and Methods. C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and with a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity and the relative values are presented as the fold-increase over the activity of the promoterless pGL3-basic vector. The length of each 5'-deletion fragment is calculated relative to the translation initiation ATG codon and is indicated to the left of each bar. The presence of putative NF-κB binding elements is also indicated. Values represent the mean ± SD of three independent experiments.
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Figure 3: Reporter analysis of the mouse CS1 and CS2 gene promoters. A. Reporter analysis of mouse CS1 gene promoter deletion constructs. B. Reporter analysis of mouse CS2 gene promoter deletion constructs. The 5'-deleted promoter segments were generated as described in Materials and Methods. C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and with a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity and the relative values are presented as the fold-increase over the activity of the promoterless pGL3-basic vector. The length of each 5'-deletion fragment is calculated relative to the translation initiation ATG codon and is indicated to the left of each bar. The presence of putative NF-κB binding elements is also indicated. Values represent the mean ± SD of three independent experiments.

Mentions: To test the minimal region required for promoter activity within the upstream CS-1 and CS-2 sequences, fragments corresponding to the regions -2554 to -144 of CS-1 and -2478 to -67 of CS-2 (relative to the ATG initiation codon), were inserted into the pGL3-basic vector and luciferase assays were performed. A significant increase in luciferase activity was observed in the C2C12 cell line (almost 20-fold and 16-fold increases following transfection with the promoter-luciferase vectors pCS1-2554/-144 and pCS2-2478/-67, respectively) compared with cells transfected with the empty vector (Fig. 3). Sequence analysis of these two promoter segments revealed that the flanking region harbours potential binding sites for multiple transcription factors including Sp1 and AP-1, in addition to the muscle specific transcription factors MEF-2 and MyoD. However, no TATA-boxes are present in these regions.


NF-kappaB mediates the transcription of mouse calsarcin-1 gene, but not calsarcin-2, in C2C12 cells.

Wang H, Yang S, Yang E, Zhu Z, Mu Y, Feng S, Li K - BMC Mol. Biol. (2007)

Reporter analysis of the mouse CS1 and CS2 gene promoters. A. Reporter analysis of mouse CS1 gene promoter deletion constructs. B. Reporter analysis of mouse CS2 gene promoter deletion constructs. The 5'-deleted promoter segments were generated as described in Materials and Methods. C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and with a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity and the relative values are presented as the fold-increase over the activity of the promoterless pGL3-basic vector. The length of each 5'-deletion fragment is calculated relative to the translation initiation ATG codon and is indicated to the left of each bar. The presence of putative NF-κB binding elements is also indicated. Values represent the mean ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828060&req=5

Figure 3: Reporter analysis of the mouse CS1 and CS2 gene promoters. A. Reporter analysis of mouse CS1 gene promoter deletion constructs. B. Reporter analysis of mouse CS2 gene promoter deletion constructs. The 5'-deleted promoter segments were generated as described in Materials and Methods. C2C12 cells were co-transfected with various promoter regions fused to firefly luciferase and with a Renilla luciferase expression control vector. The resulting firefly luciferase activity was then normalized to Renilla luciferase activity and the relative values are presented as the fold-increase over the activity of the promoterless pGL3-basic vector. The length of each 5'-deletion fragment is calculated relative to the translation initiation ATG codon and is indicated to the left of each bar. The presence of putative NF-κB binding elements is also indicated. Values represent the mean ± SD of three independent experiments.
Mentions: To test the minimal region required for promoter activity within the upstream CS-1 and CS-2 sequences, fragments corresponding to the regions -2554 to -144 of CS-1 and -2478 to -67 of CS-2 (relative to the ATG initiation codon), were inserted into the pGL3-basic vector and luciferase assays were performed. A significant increase in luciferase activity was observed in the C2C12 cell line (almost 20-fold and 16-fold increases following transfection with the promoter-luciferase vectors pCS1-2554/-144 and pCS2-2478/-67, respectively) compared with cells transfected with the empty vector (Fig. 3). Sequence analysis of these two promoter segments revealed that the flanking region harbours potential binding sites for multiple transcription factors including Sp1 and AP-1, in addition to the muscle specific transcription factors MEF-2 and MyoD. However, no TATA-boxes are present in these regions.

Bottom Line: In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1.These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells.The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gene and Cell Engineering, State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, PR China. muwhy@hotmail.com <muwhy@hotmail.com>

ABSTRACT

Background: The calsarcins comprise a novel family of muscle-specific calcineurin-interaction proteins that play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. The expression of calsarcin-1 (CS-1) is restricted to slow-twitch skeletal muscle fibres, whereas that of both calsarcin-2 (CS-2) and calsarcin-3 (CS-3) is enriched in fast-twitch fibres. However, the transcriptional control of this selective expression has not been previously elucidated.

Results: Our real-time RT-PCR analyses suggest that the expression of CS-1 and CS-2 is increased during the myogenic differentiation of mouse C2C12 cells. Promoter deletion analysis further suggests that an NF-kappaB binding site within the CS-1 promoter is responsible for the up-regulation of CS-1 transcription, but no similar mechanism was evident for CS-2. These findings are further supported by the results of EMSA analysis, as well as by overexpression and inhibition experiments in which NF-kappaB function was blocked by treatment with its inhibitor, PDTC. In addition, the overexpression of NFATc4 induces both the CS-1 and CS-2 promoters, whereas MEF2C only activates CS-1.

Conclusion: Our present data suggest that NF-kappaB is required for the transcription of mouse CS-1 but not CS-2, and that the regulation of the calsarcins is mediated also by the NFAT and MEF2 transcription factors. These results provide new insights into the molecular mechanisms governing transcription in specific muscle fibre cells. The calsarcins may also serve as a valuable mechanistic tool to better understand the regulation of calcineurin signalling during muscle differentiation.

Show MeSH
Related in: MedlinePlus