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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

Effect of Atg12 siRNA on IL-1β-induced MMP-3 activity and cell proliferation.(A) E14Tg2a ES cell-derived odontoblast-like cells were transfected with Atg12 siRNA for 24 h and treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) for either 12 h (grey bars) or 24 h (black bars), prior to carrying out an MMP-3 activity assay (left) and western blotting for Atg12, Wnt5a and MMP-3 (right). (B) BrdU-cell proliferation ELISA (for cell proliferation; graphs), BrdU Immunohistochemistry Kit (for cell proliferation; images, upper row), and APOPercentage Apoptosis Assay Kit (for apoptosis; images, 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. The BrdU ELISA kit stains the nuclei of proliferating cells dark brown. Accumulation of dye (pink-purple) denotes apoptosis in the APOPercentage kit. Images are representative of at least three independent experiments. Scale bars = 100 μm.
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pone.0124542.g006: Effect of Atg12 siRNA on IL-1β-induced MMP-3 activity and cell proliferation.(A) E14Tg2a ES cell-derived odontoblast-like cells were transfected with Atg12 siRNA for 24 h and treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) for either 12 h (grey bars) or 24 h (black bars), prior to carrying out an MMP-3 activity assay (left) and western blotting for Atg12, Wnt5a and MMP-3 (right). (B) BrdU-cell proliferation ELISA (for cell proliferation; graphs), BrdU Immunohistochemistry Kit (for cell proliferation; images, upper row), and APOPercentage Apoptosis Assay Kit (for apoptosis; images, 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. The BrdU ELISA kit stains the nuclei of proliferating cells dark brown. Accumulation of dye (pink-purple) denotes apoptosis in the APOPercentage kit. Images are representative of at least three independent experiments. Scale bars = 100 μm.

Mentions: Subsequent experiments used ES cell-derived cells (E14Tg2a) with rat KN3 cells as authentic standard odontoblast-like cells. We next employed specific siRNAs to determine whether the effects of IL-1β stimulation were specifically mediated by Atg5, LC3 or Atg12. Cells were transfected with Atg5, LC3 and Atg12 siRNAs, or a control siRNA, and were then stimulated with IL-1β as described above. These siRNAs did not affect the expression of the internal control (GAPDH) after transfection. MMP-3 mRNA was expressed only in cells transfected with control siRNA with no apparent expression in cells transfected with only the Atg5 siRNA (Fig 4A). The lack of an effect by the negative control siRNA confirmed that these IL-1β-stimulated effects were specifically mediated by Atg5. However, LC3 and Atg12 siRNAs had no effect on the expression of MMP-3, suggesting that only Atg5 is associated with IL-1β-induced MMP-3 expression in these cells (Figs 5A and 6A). Western blot analysis of Atg5, Wnt5a and MMP-3 proteins confirmed efficient silencing of the Atg5 gene (Fig 4A, right row). siRNA transfection had no effect on the expression of β-tubulin (the loading control). Atg5 siRNA caused potent suppression of Wnt5a and MMP-3 induction, indicating that Atg5, Wnt5a and MMP-3 act in similar signaling pathways, and Atg5 is upstream of Wnt5a and MMP-3 in the signaling cascade.


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)

Effect of Atg12 siRNA on IL-1β-induced MMP-3 activity and cell proliferation.(A) E14Tg2a ES cell-derived odontoblast-like cells were transfected with Atg12 siRNA for 24 h and treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) for either 12 h (grey bars) or 24 h (black bars), prior to carrying out an MMP-3 activity assay (left) and western blotting for Atg12, Wnt5a and MMP-3 (right). (B) BrdU-cell proliferation ELISA (for cell proliferation; graphs), BrdU Immunohistochemistry Kit (for cell proliferation; images, upper row), and APOPercentage Apoptosis Assay Kit (for apoptosis; images, 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. The BrdU ELISA kit stains the nuclei of proliferating cells dark brown. Accumulation of dye (pink-purple) denotes apoptosis in the APOPercentage kit. Images are representative of at least three independent experiments. Scale bars = 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403923&req=5

pone.0124542.g006: Effect of Atg12 siRNA on IL-1β-induced MMP-3 activity and cell proliferation.(A) E14Tg2a ES cell-derived odontoblast-like cells were transfected with Atg12 siRNA for 24 h and treated with IL-1β (0, 0.25, 2.5 and 25 ng/mL) for either 12 h (grey bars) or 24 h (black bars), prior to carrying out an MMP-3 activity assay (left) and western blotting for Atg12, Wnt5a and MMP-3 (right). (B) BrdU-cell proliferation ELISA (for cell proliferation; graphs), BrdU Immunohistochemistry Kit (for cell proliferation; images, upper row), and APOPercentage Apoptosis Assay Kit (for apoptosis; images, 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. The BrdU ELISA kit stains the nuclei of proliferating cells dark brown. Accumulation of dye (pink-purple) denotes apoptosis in the APOPercentage kit. Images are representative of at least three independent experiments. Scale bars = 100 μm.
Mentions: Subsequent experiments used ES cell-derived cells (E14Tg2a) with rat KN3 cells as authentic standard odontoblast-like cells. We next employed specific siRNAs to determine whether the effects of IL-1β stimulation were specifically mediated by Atg5, LC3 or Atg12. Cells were transfected with Atg5, LC3 and Atg12 siRNAs, or a control siRNA, and were then stimulated with IL-1β as described above. These siRNAs did not affect the expression of the internal control (GAPDH) after transfection. MMP-3 mRNA was expressed only in cells transfected with control siRNA with no apparent expression in cells transfected with only the Atg5 siRNA (Fig 4A). The lack of an effect by the negative control siRNA confirmed that these IL-1β-stimulated effects were specifically mediated by Atg5. However, LC3 and Atg12 siRNAs had no effect on the expression of MMP-3, suggesting that only Atg5 is associated with IL-1β-induced MMP-3 expression in these cells (Figs 5A and 6A). Western blot analysis of Atg5, Wnt5a and MMP-3 proteins confirmed efficient silencing of the Atg5 gene (Fig 4A, right row). siRNA transfection had no effect on the expression of β-tubulin (the loading control). Atg5 siRNA caused potent suppression of Wnt5a and MMP-3 induction, indicating that Atg5, Wnt5a and MMP-3 act in similar signaling pathways, and Atg5 is upstream of Wnt5a and MMP-3 in the signaling cascade.

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