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AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Maruyama Y, Arahara K, Kinoshita E, Arai K - J. Vet. Med. Sci. (2014)

Bottom Line: Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site.Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced.These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

ABSTRACT
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at -29/-17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

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Quantitative RT-PCR (qRT-PCR) analysis of the mRNA level corresponding to NF-L, NF-M.MAP2, TUBB2, TUBB3 and TUBB4a. P19 cells in the growth phase were treated with 0.5µM ATRA for 4 days to form neurospheres, and then the neurosphereswere dispersed and cultured on gelatin-coated dishes for 2 days to induce neuraldifferentiation (D). Total RNA was extracted and analyzed by qRT-PCR. The values shownare means ± SD of three independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated control cells (C).
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fig_001: Quantitative RT-PCR (qRT-PCR) analysis of the mRNA level corresponding to NF-L, NF-M.MAP2, TUBB2, TUBB3 and TUBB4a. P19 cells in the growth phase were treated with 0.5µM ATRA for 4 days to form neurospheres, and then the neurosphereswere dispersed and cultured on gelatin-coated dishes for 2 days to induce neuraldifferentiation (D). Total RNA was extracted and analyzed by qRT-PCR. The values shownare means ± SD of three independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated control cells (C).

Mentions: Enhanced TUBB4 expression in neural differentiation of P19 cells: Allneural phenotypes examined in this study increased in ATRA-treated cells. Among these, NF-L,NF-M, microtubule associated protein-2 (MAP2) and class II and class III β-tubulin wereupregulated 2- to 5-fold compared with the control. On the other hand, expression of TUBB4awas extremely increased in differentiating cells, and the mRNA level was 100-fold greaterthan that in control cells (Fig. 1).


AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Maruyama Y, Arahara K, Kinoshita E, Arai K - J. Vet. Med. Sci. (2014)

Quantitative RT-PCR (qRT-PCR) analysis of the mRNA level corresponding to NF-L, NF-M.MAP2, TUBB2, TUBB3 and TUBB4a. P19 cells in the growth phase were treated with 0.5µM ATRA for 4 days to form neurospheres, and then the neurosphereswere dispersed and cultured on gelatin-coated dishes for 2 days to induce neuraldifferentiation (D). Total RNA was extracted and analyzed by qRT-PCR. The values shownare means ± SD of three independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated control cells (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300376&req=5

fig_001: Quantitative RT-PCR (qRT-PCR) analysis of the mRNA level corresponding to NF-L, NF-M.MAP2, TUBB2, TUBB3 and TUBB4a. P19 cells in the growth phase were treated with 0.5µM ATRA for 4 days to form neurospheres, and then the neurosphereswere dispersed and cultured on gelatin-coated dishes for 2 days to induce neuraldifferentiation (D). Total RNA was extracted and analyzed by qRT-PCR. The values shownare means ± SD of three independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated control cells (C).
Mentions: Enhanced TUBB4 expression in neural differentiation of P19 cells: Allneural phenotypes examined in this study increased in ATRA-treated cells. Among these, NF-L,NF-M, microtubule associated protein-2 (MAP2) and class II and class III β-tubulin wereupregulated 2- to 5-fold compared with the control. On the other hand, expression of TUBB4awas extremely increased in differentiating cells, and the mRNA level was 100-fold greaterthan that in control cells (Fig. 1).

Bottom Line: Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site.Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced.These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

ABSTRACT
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at -29/-17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

Show MeSH
Related in: MedlinePlus