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This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors, with the approval of the Editor-in-Chief.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.

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.

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.

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

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)-1ß 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-1ß was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1ß 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-1ß-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-1ß-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells.

Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells.

Augmented EPR effect by photo-triggered tumor vascular treatment improved therapeutic efficacy of liposomal paclitaxel in mice bearing tumors with low permeable vasculature.

The effects of photo-triggered tumor vascular treatment (PVT) on the structural and functional properties of tumor vasculature were assessed in Colon-26 (C26) and B16/BL6 (B16) tumor-bearing mice. Furthermore, anti-tumor efficacy of subsequently injected PEG liposomal paclitaxel (PL-PTX) was also evaluated. As a photosensitizer, a hydrophobic porphyrin derivative was used and formulated in polymeric nanoparticle composed of polyethylene glycol-block-polylactic acid to avoid its non-specific in vivo disposition. In the mice bearing C26 with high permeable vasculature, the prominent anti-tumor activity was confirmed by PVT alone, but the subsequently injected PL-PTX did not show any additive effect. PVT itself initially induced apoptotic cell death of tumor vascular endothelial cells and platelet aggregation, which would have subsequently induced apoptosis of C26 tumor cells surrounding the vasculature. On the other hand, in the mice bearing B16 with low permeable vasculature, PVT enhanced the anti-tumor activity of subsequently injected PL-PTX, which would be attributed to the tumor disposition amount and area of PEG liposomes enhanced by PVT. These results clearly indicated that the treatment would have made it possible to provide more efficient extravasation of PL-PTX, leading to its more potent anti-tumor effect.

Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3.

A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1ß, tumor necrosis factor-α and interferon-γ) and IL-1ß-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells.