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This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors, with the approval of the Editor-in-Chief.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

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.

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.

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.

Α2 integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells.

We previously reported that interleukin 1ß acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, ß1, and ß3, integrins, we confirmed that ß1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells.

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.

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.

Differentiation of human skeletal muscle stem cells into odontoblasts is dependent on induction of α1 integrin expression.

Skeletal muscle stem cells represent an abundant source of autologous cells with potential for regenerative medicine that can be directed to differentiate into multiple lineages including osteoblasts and adipocytes. In the current study, we found that α7 integrin-positive human skeletal muscle stem cells (α7(+)hSMSCs) could differentiate into the odontoblast lineage under specific inductive conditions in response to bone morphogenetic protein-4 (BMP-4). Cell aggregates of FACS-harvested α7(+)hSMSCs were treated in suspension with retinoic acid followed by culture on a gelatin scaffold in the presence of BMP-4. Following this protocol, α7(+)hSMSCs were induced to down-regulate myogenic genes (MYOD and α7 integrin) and up-regulate odontogenic markers including dentin sialophosphoprotein, matrix metalloproteinase-20 (enamelysin), dentin sialoprotein, and alkaline phosphatase but not osteoblastic genes (osteopontin and osteocalcin). Following retinoic acid and gelatin scaffold/BMP-4 treatment, there was a coordinated switch in the integrin expression profile that paralleled odontoblastic differentiation where α1ß1 integrin was strongly up-regulated with the attenuation of muscle-specific α7ß1 integrin expression. Interestingly, using siRNA knockdown strategies revealed that the differentiation-related expression of the α1 integrin receptor positively regulates the expression of the odontoblastic markers dentin sialophosphoprotein and matrix metalloproteinase-20. These results strongly suggest that the differentiation of α7(+)hSMSCs along the odontogenic lineage is dependent on the concurrent expression of α1 integrin.

IL-1ß-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway.

We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1ß induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1ß was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1ß increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1ß-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1ß-induced proliferation of ES cell-derived odontoblast-like cells.

IL-1ß-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells.

We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1ß induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like (α7(+)hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1ß failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1ß-induced MMP-13 expression is linked to this IL-1ß-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1ß-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7(+)hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7(+)hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1ß-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7(+)hSMSC-OB cells are regulated by ADAM-28.

Site-specific incorporation of functional components into RNA by an unnatural base pair transcription system.

Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3'-terminus. We found that the efficient incorporation into a position close to the 3'-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.