Retraction notice to "Gelatin scaffold combined with bone morphogenetic protein-4 induces odontoblast-like cell differentiation involving integrin profile changes, autophagy-related gene 10, and Wnt5 sequentially in human induced pluripotent stem cells" [Differentiation 93 (2017) 1-14].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors, with the approval of the Editor-in-Chief. The journal was initially contacted by the corresponding author to report the unavailability of the raw data of the results presented by the article, as well as the similarity between the Western blots from Figure 11A (MMP-3) and Figure 11C (MMP-3). Also, a significant amount of text has been reused from the articles that the authors have previously published in the Experimental Cell Research 341 (2016) 92-104 https://doi.org/10.1016/j.yexcr.2016.01.010 and the Journal of Biological Chemistry 289 (2014) 14380-14391 https://doi.org/10.1074/jbc.M113.526772. All of the authors except Nobuaki Ozeki and Taiki Hiyama have reportedly agreed to retract the article. N. Ozeki left Aichi Gakuin University in March 2018 and does not respond to co-authors inquiries, while T. Hiyama left Aichi Gakuin University and could not be reached. The authors deeply regret this error and any inconvenience it may have caused.

MicroRNA-211 and autophagy-related gene 14 signaling regulate osteoblast-like cell differentiation of human induced pluripotent stem cells.

MicroRNAs (miRNAs) have been the subject of recent attention as key regulatory factors in cell differentiation. In the current study, to explore the early signaling cascade of osteogenic differentiation of human induced pluripotent stem (hiPS) cells, we investigated miR-211 regulation and autophagy-related gene (Atg) signaling in osteogenic differentiation. In addition to reciprocal strong induction of miR-211 expression in differentiated cells following osteogenic differentiation, we found abundant Argonaute 3 bound to miR-211. There were also dramatic increases in the mRNA and protein levels of Atg14 together with increases in the amount of autophagosomes as well as autophagic fluxes. While transfection of a miR-211 inhibitor abrogated the induction of Atg14, autophagy events, osteoblast differentiation markers, and induction of calcification were suppressed markedly. Treatment with small interfering RNAs against Atg14 also suppressed the osteogenic differentiation medium (ODM)-induced increase in osteogenic differentiation. The osteogenic phenotype was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy inducer). Taken together with our previous findings, we have revealed a unique sequential cascade involving miR-211 and Atg14 in ODM-induced differentiation of hiPS cells into osteoblast-like cells at a relatively early stage.

Gelatin scaffold combined with bone morphogenetic protein-4 induces odontoblast-like cell differentiation involving integrin profile changes, autophagy-related gene 10, and Wnt5 sequentially in human induced pluripotent stem cells.

While human induced pluripotent stem (hiPS) cells have potential use in regenerative medicine, there are no reports on odontoblastic differentiation of hiPS cells. In the current study, to examine integrin profiles and explore the early signaling cascade of odontoblastic differentiation in hiPS cells, we investigated the regulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling in gelatin scaffold (GS) combined with bone morphogenetic protein (BMP)-4 (GS/BMP-4)-mediated odontoblastic differentiation. Following GS/BMP-4 treatment, there was a dramatic loss of α3 and α6 integrins, and reciprocal strong induction of α1 integrin expression in the differentiated cells. GS/BMP-4 increased the mRNA and protein levels of Atg10, Lrp5/Fzd9 (an Atg10 receptor), and Wnt5 together with the amount of autophagosomes and autophagic fluxes. Treatment with siRNAs against Atg10 and Wnt5a individually suppressed the GS/BMP-4-induced increase in odontoblastic differentiation. The odontoblastic phenotype was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have replicated our results from the rodent system in a novel human system. We have revealed a unique sequential cascade involving Atg10, Wnt5a, α1 integrin, and matrix metalloproteinase-3 in GS/BMP-4-induced differentiation of hiPS cells into odontoblast-like cells at a relatively early stage.

Polyphosphate-induced matrix metalloproteinase-13 is required for osteoblast-like cell differentiation in human adipose tissue derived mesenchymal stem cells.

Inorganic polyphosphate [Poly(P)] induces differentiation of osteoblastic cells. In this study, matrix metalloproteinase (MMP)-13 small interfering RNA (siRNA) was transfected into human adipose tissue-derived mesenchymal stem cells (hAT-MSC) to investigate whether MMP-13 activity induced by Poly(P) is associated with osteogenic differentiation. Real-time quantitative polymerase chain reaction, Western blotting, and an MMP-13 activity assay were used in this study. Poly(P) enhanced expression of mature osteoblast markers, such as osteocalcin (BGLAP) and osteopontin (SPP1), osterix (OSX), and bone sialoprotein (BSP), and increased alkaline phosphatase (ALP) activity and calcification capacity in hAT-MSCs. These cells also developed an osteogenic phenotype with increased expression of Poly(P)-induced expression of MMP-13 mRNA and protein, and increased MMP-13 activity. MMP-13 siRNA potently suppressed the expression of osteogenic biomarkers BGLAP, SPP1, OSX, BSP, and ALP, and blocked osteogenic calcification. Taken together, Poly(P)-induced MMP-13 regulates differentiation of osteogenic cells from hAT-MSCs.

Bone morphogenetic protein-induced cell differentiation involves Atg7 and Wnt16 sequentially in human stem cell-derived osteoblastic cells.

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like cells with bone morphogenetic protein (BMP)-2. To explore the early signaling cascade for osteoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling during BMP-2-mediated human osteoblastic differentiation. In a screening experiment, BMP-2 increased the mRNA and protein levels of Atg7, Wnt16, and Lrp5/Fzd2 (a Wnt receptor), but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, Atg5, Atg12, Wnt3a, or Wnt5, together with the amounts of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg7 and Wnt16 individually suppressed the BMP-2-induced increase in osteoblastic differentiation. The osteoblastic phenotype, involving osteocalcin (BGLAP), osteopontin (SPP1), and osterix (SP7) expression, decreased when autophagy was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade of BMP-2→Atg7→Wnt16→Lrp5/Fzd2→matrix metalloproteinase-13→osteoblastic differentiation. This cascade results in a potent increase in osteoblastic cell differentiation, indicating the unique involvement of Atg7, autophagy, and Wnt16 signaling in BMP-2-induced differentiation of α7(+)hSMSCs into osteoblast-like cells at a relatively early stage.

Wnt16 Signaling Is Required for IL-1ß-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells.

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1ß induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1ß was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1ß increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1ß-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1ß→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1ß-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.

Autophagy-related gene 5 and Wnt5 signaling pathway requires differentiation of embryonic stem cells into odontoblast-like cells.

We previously confirmed a unique and unanticipated role for an α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and matrix metalloproteinase (MMP)-3-mediated signaling cascade, in driving the odontoblast-like differentiation of mouse embryonic stem (ES) cells in a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). To explore the early signaling cascade for odontoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and Wnt signaling by CS/BMP-4 mediated odontoblast differentiation. In a screening experiment, CS/BMP-4 increased the mRNA and protein levels of Atg5, Lrp5/Fzd9 (an Atg5 receptor), and Wnt5, but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, and Atg12, together with the amount of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg5 and Wnt5 individually suppressed the CS/BMP-4-induced increase in odontoblast differentiation. The odontoblastic phenotype, involving dentin matrix protein-1 and dentin sialophosphoprotein expression, decreased when autophagy was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade involving Atg5, Wnt5a, α2 integrin, Emmprin, and MMP-3. This cascade results in a potent increase in odontoblastic cell differentiation, indicating the unique involvement of Atg5, autophagy and Wnt5 signaling in CS/BMP-4-induced differentiation of ES cells into odontoblast-like cells, at a relatively early stage.

RETRACTED: New findings for dentin sialophosphoprotein studies: Applications of purified odontoblast-like cells derived from stem cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Authors. After the retraction of the article [Hiyama T, Ozeki N, Mogi M, Yamaguchi H, Kawai R, Nakata K, Kondo A, Nakamura H. 2013. Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells. PLoS ONE 8(12): e83563. doi:10.1371/journal.pone.0083563] which contained fabricated/falsified data, the authors attempted to confirm original data for the results presented in their related publications. As a result, they reached a conclusion that there were no original data for the results presented in several their publications. This article was written on the basis of the seven publications retracted or to be retracted and it is no longer reliable. Reference 24: N. Ozeki, M. Mogi, R. Kawai, H. Yamaguchi, T. Hiyama, K. Nakata, H. Nakamura Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin PLoS One, 8 (2013), p. e80026 Reference 25:R. Kawai, N. Ozeki, H. Yamaguchi, T. Tanaka, K. Nakata, M. Mogi, H. Nakamura Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method Oral Dis, 20 (2014), pp. 395-403 Reference 26:N. Ozeki, M. Mogi, H. Yamaguchi, T. Hiyama, R. Kawai, N. Hase, K. Nakata, H. Nakamura, R.H. Kramer Differentiation of human skeletal muscle stem cells into odontoblasts is dependent on induction of alpha1 integrin expression J Biol Chem, 289 (2014), pp. 14380-14391 Reference 42:N. Ozeki, N. Hase, R. Kawai, H. Yamaguchi, T. Hiyama, A. Kondo, K. Nakata, M. Mogi Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3 Exp Cell Res, 331 (2015), pp. 105-114 Reference 43: N. Ozeki, N. Hase, H. Yamaguchi, T. Hiyama, R. Kawai, A. Kondo, K. Nakata, M. Mogi Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells Exp Cell Res, 333 (2015), pp. 303-315 Reference 44: N. Ozeki, R. Kawai, N. Hase, T. Hiyama, H. Yamaguchi, A. Kondo, K. Nakata, M. Mogi Alpha2 integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like Exp Cell Res, 331 (2015), pp. 21-37 Reference 45: N. Ozeki, M. Mogi, N. Hase, T. Hiyama, H. Yamaguchi, R. Kawai, A. Kondo, T. Matsumoto, K. Nakata Autophagy-related gene 5 and Wnt5 signaling pathway requires differentiation of embryonic stem cells into odontoblast-like cells Exp Cell Res, 341 (2016), pp. 92-104 All of the authors except Nobuaki Ozeki have agreed to retract the article. Nobuaki Ozeki, the corresponding author and the first author of the article, left Aichi Gakuin University in March 2018, and does not respond to co-authors inquiries. The authors deeply regret this error and any inconvenience it may have caused.

Products of dentin matrix protein-1 degradation by interleukin-1ß-induced matrix metalloproteinase-3 promote proliferation of odontoblastic cells.

We have previously reported that interleukin (IL)-1ß induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in mouse embryonic stem cell (ESC)-derived odontoblast-like cells, suggesting that MMP-3 plays a potentially unique physiological role in regeneration by odontoblast-like cells. MMPs are able to process virtually any component of the extracellular matrix, including collagen, laminin and bioactive molecules. Because odontoblasts produce dentin matrix protein-1 (DMP-1), we examined whether the degraded products of DMP-1 by MMP-3 contribute to enhanced proliferation in odontoblast-like cells. IL-1ß increased mRNA and protein levels of odontoblastic marker proteins, including DMP-1, but not osteoblastic marker proteins, such as osteocalcin and osteopontin. The recombinant active form of MMP-3 could degrade DMP-1 protein but not osteocalcin and osteopontin in vitro. The exogenous degraded products of DMP-1 by MMP-3 resulted in increased proliferation of odontoblast-like cells in a dose-dependent manner. Treatment with a polyclonal antibody against DMP-1 suppressed IL-1ß-induced cell proliferation to a basal level, but identical treatment had no effect on the IL-1ß-induced increase in MMP-3 expression and activity. Treatment with siRNA against MMP-3 potently suppressed the IL-1ß-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Similarly, treatment with siRNAs against Wnt5a and Wnt5b suppressed the IL-1ß-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Rat KN-3 cells, representative of authentic odontoblasts, showed similar responses to the odontoblast-like cells. Taken together, our current study demonstrates the sequential involvement of Wnt5, MMP-3, DMP-1 expression, and DMP-1 degradation products by MMP-3, in effecting IL-1ß-induced proliferation of ESC-derived odontoblast-like cells.

Polyphosphate-induced matrix metalloproteinase-3-mediated proliferation in rat dental pulp fibroblast-like cells is mediated by a Wnt5 signaling cascade.

Although it is known that inorganic polyphosphate [Poly(P)] induces differentiation of osteoblasts, there are few reports concerning its effects on cell proliferation, especially in fibroblasts. Because we found that Poly(P) stimulates the proliferation of purified rat dental pulp fibroblast-like cells (DPFCs), matrix metalloproteinase (MMP)-3 small interfering RNA (siRNA) was transfected into purified rat DPFCs to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation in DPFCs. Real-time quantitative polymerase chain reaction, Western blots, an MMP-3 activity assay, and an enzyme-linked immunosorbent assay to assess cell proliferation were used in this study. Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity and cell proliferation. Silencing of MMP-3 expression with siRNA yielded potent and significant suppression of Poly(P)-induced MMP-3 expression and activity, and decreased cell proliferation. Poly(P) also increased mRNA and protein levels of Wnt5 and the Wnt receptor Lrp5/Fzd9. Although exogenous MMP-3 could not induce Wnt5, exogenous Wnt5 was found to increase MMP-3 activity and, interestingly, the proliferation rate of DPFCs. Transfection with Wnt5a siRNA suppressed the Poly(P)-induced increase in MMP-3 expression and suppressed cell proliferation. These results demonstrate the sequential involvement of Wnt5 and MMP-3 in Poly(P)-induced proliferation of DPFCs, and may have relevance in our understanding and ability to improve wound healing following dental pulp injury.