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Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

Ozeki N, Hase N, Hiyama T, Yamaguchi H, Kawai R, Kondo A, Matsumoto T, Nakata K, Mogi M - PLoS ONE (2015)

Bottom Line: Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state.IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12.Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi, 464-8651, Japan.

ABSTRACT
We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

No MeSH data available.


Related in: MedlinePlus

Determination of the signaling sequence using specific siRNAs.(A–F) Cells were incubated in serum-free medium for 24 h and then treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) and Atg5, Wnt5a, or MMP-3 siRNAs. mRNA expression levels of Atg5, Wnt5a and MMP-3 relative to the control (18S rRNA) were determined by qPCR and western blotting for LC3, Atg5, Wnt5a, and MMP-3 (lower row). β-tubulin was used as a housekeeping protein in western blots. **P < 0.01 vs. control; ##P < 0.01 vs. control siRNA; †P < 0.01, as indicated by the bracket.
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pone.0124542.g007: Determination of the signaling sequence using specific siRNAs.(A–F) Cells were incubated in serum-free medium for 24 h and then treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) and Atg5, Wnt5a, or MMP-3 siRNAs. mRNA expression levels of Atg5, Wnt5a and MMP-3 relative to the control (18S rRNA) were determined by qPCR and western blotting for LC3, Atg5, Wnt5a, and MMP-3 (lower row). β-tubulin was used as a housekeeping protein in western blots. **P < 0.01 vs. control; ##P < 0.01 vs. control siRNA; †P < 0.01, as indicated by the bracket.

Mentions: Using several specific siRNAs, we examined the sequential order of Atg5, Wnt5 and MMP-3 expression in odontoblast-like cells by qPCR and western blot analysis (Fig 7A–7F). IL-1β-induced expression of Atg5 was only inhibited by Atg5 siRNA (Fig 7), whereas Wnt5a expression was inhibited by both Wnt5a and Atg5 siRNAs (Fig 7). Furthermore, IL-1β-induced MMP-3 expression was inhibited by Atg5, Wnt5a and MMP-3 siRNAs (Fig 7). Similar results were obtained from western blot analysis (Fig 7). Thus, taken together with the above data, this signaling cascade appears to be IL-1β→Atg5→Wnt5a→MMP-3 and is intimately involved in the proliferation of stem cell-derived odontoblast-like cells.


Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

Ozeki N, Hase N, Hiyama T, Yamaguchi H, Kawai R, Kondo A, Matsumoto T, Nakata K, Mogi M - PLoS ONE (2015)

Determination of the signaling sequence using specific siRNAs.(A–F) Cells were incubated in serum-free medium for 24 h and then treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) and Atg5, Wnt5a, or MMP-3 siRNAs. mRNA expression levels of Atg5, Wnt5a and MMP-3 relative to the control (18S rRNA) were determined by qPCR and western blotting for LC3, Atg5, Wnt5a, and MMP-3 (lower row). β-tubulin was used as a housekeeping protein in western blots. **P < 0.01 vs. control; ##P < 0.01 vs. control siRNA; †P < 0.01, as indicated by the bracket.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403923&req=5

pone.0124542.g007: Determination of the signaling sequence using specific siRNAs.(A–F) Cells were incubated in serum-free medium for 24 h and then treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) and Atg5, Wnt5a, or MMP-3 siRNAs. mRNA expression levels of Atg5, Wnt5a and MMP-3 relative to the control (18S rRNA) were determined by qPCR and western blotting for LC3, Atg5, Wnt5a, and MMP-3 (lower row). β-tubulin was used as a housekeeping protein in western blots. **P < 0.01 vs. control; ##P < 0.01 vs. control siRNA; †P < 0.01, as indicated by the bracket.
Mentions: Using several specific siRNAs, we examined the sequential order of Atg5, Wnt5 and MMP-3 expression in odontoblast-like cells by qPCR and western blot analysis (Fig 7A–7F). IL-1β-induced expression of Atg5 was only inhibited by Atg5 siRNA (Fig 7), whereas Wnt5a expression was inhibited by both Wnt5a and Atg5 siRNAs (Fig 7). Furthermore, IL-1β-induced MMP-3 expression was inhibited by Atg5, Wnt5a and MMP-3 siRNAs (Fig 7). Similar results were obtained from western blot analysis (Fig 7). Thus, taken together with the above data, this signaling cascade appears to be IL-1β→Atg5→Wnt5a→MMP-3 and is intimately involved in the proliferation of stem cell-derived odontoblast-like cells.

Bottom Line: Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state.IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12.Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi, 464-8651, Japan.

ABSTRACT
We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

No MeSH data available.


Related in: MedlinePlus