It is known that Wnt/β-catenin signaling induces endochondral ossification and plays a significant role in the pathophysiology of osteoarthritis (OA). Sclerostin is a potent inhibitor of the Wnt/β-catenin signaling pathway. This study investigated the role of sclerostin in the endochondral differentiation under an OA-like condition induced by proinflammatory cytokines. ATDC5 cells were used to investigate chondrogenic differentiation and terminal calcification, and 10 ng/ml IL-1β and/or 200 ng/ml sclerostin were added to the culture medium. IL-1β impaired early chondrogenesis from undifferentiated state into proliferative chondrocytes, and it was not altered by sclerostin. IL-1β induced progression of chondrogenic differentiation in the late stage and promoted terminal calcification. These processes were inhibited by sclerostin and chondrogenic phenotype was restored. In addition, sclerostin restored IL-1β-induced upregulation of Wnt/β-catenin signaling in the late stage. This study provides insights into the possible role of sclerostin in the chondrogenic differentiation under the IL-1β-induced OA-like environment. Suppression of Wnt signaling by an antagonist may play a key role in the maintenance of articular homeostasis and has a potential to prevent the progression of OA. Thus, sclerostin is a candidate treatment option for OA. Osteoarthritis (OA) is a degenerative joint disease, characterized by cartilage degradation, subchondral bone sclerosis, osteophyte formation, and synovial inflammation. A complex network of multifactorial mechanisms including biochemical, mechanical, and enzymatic aspects are involved in the pathogenesis of OA . ProinflThe Wnt/β-catenin signaling pathway plays a significant role in the pathophysiology of OA . A previSOST gene, is known to be one of the Wnt signaling antagonists Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: NoReviewer #3: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this manuscript, the authors use the ATDC5 cell line to examine the dual effects of sclerostin and interleukin 1 beta on cell differentiation, cell mineralisation and gene expression of chondrocyte markers and components of the wnt signalling pathway. They found that IL-1B impaired chondrocyte differentiation, and that the addition of sclerostin had no additional effect on this. IL-1B also drove chondrocyte mineralisation and sclerostin was able to restore this.1) I am unclear as to the novelty of this work, as the dual effects of sclerostin and inflammatory cyotkines have been previously investigated .2) I have concerns with some of the data - in particular the alizarin red staining  which appear to have staining outside of the well and therefore make me question these results. Higher power is also needed to confirm this is not ectopic calcification. Further, some of the stats are questionable - e.g. Fig 3B VEGF has huge error bars which are significant?3) Some of the statements in the results are incorrect e.g. line 118 - only one marker was significantly changed and this statement suggests all were. This needs rectifying throughout4) Were other markers of endochondral ossificaion examined - e.g. PTH/PTHrP/Ihh? The authors looked at ADAMTS5, but what about TIMPs?5) Justification of concentrations used requiredReviewer #2: In this work, Miyatake and colleagues provide a straightforward study showing the potential of sclerostin to inhibit canonical Wnt signaling and be a therapeutic target for osteoarthritis (OA). This study provides incremental knowledge of sclerostin’s role in an IL1-β induced OA in vitro model. I am not convinced that the cell line used in this study is the best model to look at early and later stages of chondrogenic differentiation in the context of endochondral ossification, especially as these are teratoma-derived if I understand correctly. The authors need to speak to why this cell line was used. Below are additional comments to the authors:Introduction- Would be beneficial to reference what is known from in vivo models of sclerostin and canonical Wnt pathway components, specifically knock-out or transgenic mouse models, to show physiological, whole system effects.Methods- Need to provide more detail about the source/company, species, and phenotype of the ATDC5 cells.- Concerned about cells cultured through 7 weeks in 2D and the environment affecting phenotype more than the pharmacological treatment- Line 63, clarify that for early stage treatment was started at 24 hours after confluency and for late stage, treatment was started on day 21 as the wording right in this current draft is confusing.- Why standard error of the mean vs standard deviation for plots?- Need to explain and/or reference why 10 ng/mL IL-1β was used to induce inflammation vs. other concentrations.- Need a control group of cells that have not been subjected to chondrogenic media to characterize and compare how these cells behave over the treatment period in 2D culture.- Would be helpful to look at protein levels (immunostaining if not westerns) of active beta-catenin and downstream targets (e.g. Axin 1/2) to further confirm sclerostin’s effects.Discussion- Would be beneficial to provide commentary on how this knowledge could be used for treatment in diseases such as osteoarthritis, in context with this in vitro model compared to in vivo models.Reviewer #3: This manuscript reports that sclerostin prevents late stage of chondrogenic differentiation induced by interleukin-1ß in ATDC5 cells through inhibition of Wnt/ß-catenin signaling. The authors show that sclerostin has no effect on the expression of chondrogenic differentiation markers and Wnt/ß-catenin pathway genes in response to interleukin-1ß at early stage . However, sclerostin counteracts the effect of interleukin-1ß on the expression of most these genes at late stage . Although this study indicates an antagonistic effect of sclerostin on interleukin-1ß in chondrogenic differentiation, the present results are too preliminary to warrant a publication.1. Interleukin-1ß treatment increases the expression of some Wnt/ß-catenin genes, and inhibits the expression of others , but the extent of increase or decrease is very limited for most genes. What would be the overall outcome of these regulations on Wnt/ß-catenin signaling? It is necessary to assess whether Wnt/signaling is indeed increased in the cells treated interleukin-1ß. Similarly, the authors should also examine whether sclerostin exerts a net effect on Wnt/signaling in cells treated by interleukin-1ß.2. It is not clear how sclerostin regulates the expression of these genes in the absence of interleukin-1ß. It inhibits the expression of Wnt/ß-catenin pathway genes directly or indirectly by inhibiting bone formation? Sclerostin is a secreted protein that antagonizes Wnt signaling by binding to LRP5/6, how it inhibits axin1 and ctnb-1 (ß-catenin) expression, is there a positive feedback of Wnt/ß-catenin signaling in the cells at this stage of chondrogenic differentiation.3. The effect of sclerostin on BMP signaling needs to be examined to see if it inhibits chondrogenic differentiation also by antagonizing this pathway.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  24 Jul 2020Please find in the following paragraphs a point-by-point reply to the comments of the reviewers.Reviewer #1: In this manuscript, the authors use the ATDC5 cell line to examine the dual effects of sclerostin and interleukin 1 beta on cell differentiation, cell mineralisation and gene expression of chondrocyte markers and components of the wnt signalling pathway. They found that IL-1B impaired chondrocyte differentiation, and that the addition of sclerostin had no additional effect on this. IL-1B also drove chondrocyte mineralisation and sclerostin was able to restore this.1) I am unclear as to the novelty of this work, as the dual effects of sclerostin and inflammatory cyotkines have been previously investigated .Response: The strong point of this study is the investigation of these effects through whole process of chondrogenic differentiation from mesenchymal chondroprogenitor to terminal calcification. Especially, the role of inflammatory cyotkines and sclerostin in the terminal calcification of chondrocyte has not been investigated in the previous studies, which it is considered as the novelty of the present study. Additional comments were described in the discussion (Line 186-198).2) I have concerns with some of the data - in particular the alizarin red staining  which appear to have staining outside of the well and therefore make me question these results. Higher power is also needed to confirm this is not ectopic calcification. Further, some of the stats are questionable - e.g. Fig 3B VEGF has huge error bars which are significant?Response: The images of alizarin red and alcian blue were replaced with new ones. Photomicrographs with high power magnification were added to the data. Relative mRNA expression of VEGF was re-analyzed by real-time PCR, and the data were replaced in Fig. 3B.3) Some of the statements in the results are incorrect e.g. line 118 - only one marker was significantly changed and this statement suggests all were. This needs rectifying throughoutResponse: We apologize this incorrect writing. The description ‘The mRNA expressions of markers for cartilage catabolism were’ was revised to ‘The mRNA expression of MMP-13, one of markers for cartilage catabolism, was’ (Line 142-143).4) Were other markers of endochondral ossificaion examined - e.g. PTH/PTHrP/Ihh? The authors looked at ADAMTS5, but what about TIMPs?Response: Unfortunately, the other markers including PTH/PTHrP/Ihh and TIMPs were not investigated. We would like to analyze them in the future opportunity.5) Justification of concentrations used requiredResponse: IL-1β was not cytotoxic up to a concentration of 100 ng/mL . Of these concentrations, 10 ng/mL IL-1β is considered as an optimized concentration to induce OA-like condition in vitro, and this concentration was also used in most of previous studies . Text was revised (Line 217- 219) and following references were added.12. Graeser AC, et al. Synergistic chondroprotective effect of alpha-tocopherol, ascorbic acid, and selenium as well as glucosamine and chondroitin on oxidant induced cell death and inhibition of matrix metalloproteinase-3--studies in cultured chondrocytes. Molecules. 2009; 15: 27-39.13. Shi S, et al. Silencing of Wnt5a prevents interleukin-1beta-induced collagen type II degradation in rat chondrocytes. Exp Ther Med. 2016; 12: 3161-3166.14. Wang F, et al. IL-1beta receptor antagonist (IL-1Ra) combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in rat and human osteoarthritis chondrocytes. Arthritis Res Ther. 2019; 21: 171.Reviewer #2: In this work, Miyatake and colleagues provide a straightforward study showing the potential of sclerostin to inhibit canonical Wnt signaling and be a therapeutic target for osteoarthritis (OA). This study provides incremental knowledge of sclerostin’s role in an IL1-β induced OA in vitro model. I am not convinced that the cell line used in this study is the best model to look at early and later stages of chondrogenic differentiation in the context of endochondral ossification, especially as these are teratoma-derived if I understand correctly. The authors need to speak to why this cell line was used.Response: The following description was added to the revised manuscript (Line 186-198).As described in detail previously , ATDC5 is a good model system for studying the dynamic processes of chondrogenesis, and many findings in this system may have relevance to chondrogenesis in vivo. In our previous study, the role of sclerostin as an inhibitor of the canonical Wnt signaling pathway in the chondrogenic differentiation could be characterized using the same model system [11]. This established model system was used to investigate the effects on chondrogenic differentiation at the different timing of early and later stages in the present study. Although several in vivo OA models have been developed to investigate the pathological feature and therapeutic effects, they need to be considered influence under the multifactorial and complex conditions including mechanical load, synovial inflammation, cartilage degeneration and abnormal bone remodeling. This study simply focused the effect of sclerostin on the cytokine among several factors associated with OA pathogenesis.Below are additional comments to the authors:Introduction- Would be beneficial to reference what is known from in vivo models of sclerostin and canonical Wnt pathway components, specifically knock-out or transgenic mouse models, to show physiological, whole system effects.Response: Descriptions about in vivo models of sclerostin and canonical Wnt pathway components were added to the introduction section .Methods- Need to provide more detail about the source/company, species, and phenotype of the ATDC5 cells.Response: Accordingly, description about ATDC5 was added to the text (Line 58-60).- Concerned about cells cultured through 7 weeks in 2D and the environment affecting phenotype more than the pharmacological treatmentResponse: Since it is difficult to create the model for terminal calcification in 3D culture of chondrocyte, we used 2D culture model. Addition of IL-1β accelerated this phenotypic change, but treatment with sclerostin showed significant effects of restoration even in this environment.- Line 63, clarify that for early stage treatment was started at 24 hours after confluency and for late stage, treatment was started on day 21 as the wording right in this current draft is confusing.Response: The description was revised as suggested (Line 73-75).- Why standard error of the mean vs standard deviation for plots?Response: It was our mistake. All data were given as mean and standard deviation. The statement was revised (Line 130).- Need to explain and/or reference why 10 ng/mL IL-1β was used to induce inflammation vs. other concentrations.Response: IL-1β was not cytotoxic up to a concentration of 100 ng/mL . Of these concentrations, 10 ng/mL IL-1β is considered as an optimized concentration to induce OA-like condition in vitro, and this concentration was also used in most of previous studies . Text was revised and following references were added (Line 217- 219).12. Graeser AC, et al. Synergistic chondroprotective effect of alpha-tocopherol, ascorbic acid, and selenium as well as glucosamine and chondroitin on oxidant induced cell death and inhibition of matrix metalloproteinase-3--studies in cultured chondrocytes. Molecules. 2009; 15: 27-39.13. Shi S, et al. Silencing of Wnt5a prevents interleukin-1beta-induced collagen type II degradation in rat chondrocytes. Exp Ther Med. 2016; 12: 3161-3166.14. Wang F, et al. IL-1beta receptor antagonist (IL-1Ra) combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in rat and human osteoarthritis chondrocytes. Arthritis Res Ther. 2019; 21: 171.- Need a control group of cells that have not been subjected to chondrogenic media to characterize and compare how these cells behave over the treatment period in 2D culture.Response: To characterize the cells that are not subjected to chondrogenic media, ATDC5 cells were also cultured without ITS. Chondrogenic differentiation with proteoglycan synthesis was confirmed by positive staining with Alcian blue after 3 weeks . Less intense staining was observed without ITS. Terminal calcification was confirmed by positive staining with Alizarin red after 7 weeks . Less staining was identified without ITS.- Would be helpful to look at protein levels (immunostaining if not westerns) of active beta-catenin and downstream targets (e.g. Axin 1/2) to further confirm sclerostin’s effects.Response: Accordingly, immunostaining for ß-catenin, Axin1, and Axin 2 was performed and data were added .Discussion- Would be beneficial to provide commentary on how this knowledge could be used for treatment in diseases such as osteoarthritis, in context with this in vitro model compared to in vivo models.Response: Following comment was added to the revised text (Line 193-198).Although several in vivo OA models have been developed to investigate the pathological feature and therapeutic effects, they need to be considered influence under the multifactorial and complex conditions including mechanical load, synovial inflammation, cartilage degeneration and abnormal bone remodeling. This study simply focused the effect of sclerostin on the cytokine among several factors associated with OA pathogenesis.Reviewer #3: This manuscript reports that sclerostin prevents late stage of chondrogenic differentiation induced by interleukin-1ß in ATDC5 cells through inhibition of Wnt/ß-catenin signaling. The authors show that sclerostin has no effect on the expression of chondrogenic differentiation markers and Wnt/ß-catenin pathway genes in response to interleukin-1ß at early stage . However, sclerostin counteracts the effect of interleukin-1ß on the expression of most these genes at late stage . Although this study indicates an antagonistic effect of sclerostin on interleukin-1ß in chondrogenic differentiation, the present results are too preliminary to warrant a publication.1. Interleukin-1ß treatment increases the expression of some Wnt/ß-catenin genes, and inhibits the expression of others , but the extent of increase or decrease is very limited for most genes. What would be the overall outcome of these regulations on Wnt/ß-catenin signaling? It is necessary to assess whether Wnt/signaling is indeed increased in the cells treated interleukin-1ß. Similarly, the authors should also examine whether sclerostin exerts a net effect on Wnt/signaling in cells treated by interleukin-1ß.Response: As reviewer suggested, effects of interleukin-1ß on LRP5/6 seems to be limited. However, expression of phosphorylated LRP6 was increased by interleukin-1ß . Gene expression of Axin2 was re-examined and data showed the same trend as the other related genes. We apologize the data in the initial submission were not correct due to technical error and the graph was replaced in the revised figure. In addition, we assessed the expression of SOST in the cells during chondeogenic differentiation with or without Interleukin-1ß . Interleukin-1ß significantly suppressed the expression of SOST. This is thought to affect indirectly upregulation of Wn/ß-catenin signaling. Similar mechanism was reported by Yoshida et al . Interleukin-1ß activated Wnt/ß-catenin signaling by suppression of Wnt antagonist, DKK-1.2. It is not clear how sclerostin regulates the expression of these genes in the absence of interleukin-1ß. It inhibits the expression of Wnt/ß-catenin pathway genes directly or indirectly by inhibiting bone formation? Sclerostin is a secreted protein that antagonizes Wnt signaling by binding to LRP5/6, how it inhibits axin1 and ctnb-1 (ß-catenin) expression, is there a positive feedback of Wnt/ß-catenin signaling in the cells at this stage of chondrogenic differentiation.Response: Our previous study  demonstrated that sclerostin directly inhibited the expression of Wnt/ß-catenin pathway genes with downregulation of LRP5/6 during chondrogenic differentiation in the absence of interleukin-1ß. When added to the interleukin-1ß, sclerostin suppressed the expressions of LRP6 and phosphorylated LRP6 in the present study. It is unclear whether there is a positive feedback of Wnt/ß-catenin signaling in the cells.3. The effect of sclerostin on BMP signaling needs to be examined to see if it inhibits chondrogenic differentiation also by antagonizing this pathway.Response: The mRNA expression level of BMP-2 was additionally investigated and added to ta data . BMP2 was upregulated by interleukin-1ß, and it was inhibited by sclerostin.Attachmentresponse to reviewers_200721.docxSubmitted filename: Click here for additional data file. 12 Aug 2020PONE-D-19-33198R1Sclerostin inhibits interleukin-1β-induced late stage chondrogenic differentiation through downregulation of Wnt/β-catenin signaling pathwayPLOS ONEDear Dr. Kumagai,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Sep 26 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: (No Response)Reviewer #3: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: I Don't KnowReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: The authors have addressed all of the original comments and critiques except the issue with standard deviation vs. standard error of the mean. In the comments to reviewer, the authors comment that standard deviation was calculated and used to show variance in the data sets. However, the text reads "standard error" which is not standard deviation - as it is the standard deviation divided by the square root of the sample size. Please correct accurately and update in the text.Reviewer #3: This revised manuscript includes additional experiments examining the effects of IL-1ß and sclerostin on the expression of Wnt/ß-catenin signaling components at early and late stages of chondrogenic differentiation in ATDC5 cells. It appears that IL-1ß inhibits the expression of both positive and negative regulators of Wnt/ß-catenin signaling at early stages of chondrogenic differentiation, and promotes their expression in terminal calcification at late stages of chondrogenic differentiation. In addition, sclerostin counteracts the effects of IL-1ß at late stages but not at early stages. However, the present version failed to address the issues raised in my previous review. In particular, it remains unclear whether IL-1ß inhibits Wnt/ß-catenin signaling at early stages of chondrogenic differentiation, and promotes Wnt/ß-catenin signaling at late stages of chondrogenic differentiation.I understand that IL-1ß may exert differential effects at early and late stages of chondrogenic differentiation. Nevertheless, IL-1ß seems to regulate the expression of both positive and negative regulators of Wnt/ß-catenin signaling. Thus, the net outcome on Wnt/ß-catenin signaling is unclear.The effect of sclerostin on IL-1ß at late stages of chondrogenic differentiation is likely indirect. Sclerostin binds to LRP5/6 to inhibit Wnt/ß-catenin signaling, and it is believed that the antagonistic effect of sclerostin on bone formation is mediated by Wnt signaling. Thus, there is no sufficient novelty in the present study.It is difficult to understand how the experiments were done, in particular, when the early effects were analyzed after treatment. For example, the results presented in Fig. 1C seem to be obtained after 3 weeks, but it is not clear when the cells were treated, and they represent early or late effect?**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Sep 2020Please find in the following paragraphs a point-by-point reply to the comments of the reviewers.Reviewer #1: (No Response)Reviewer #2: The authors have addressed all of the original comments and critiques except the issue with standard deviation vs. standard error of the mean. In the comments to reviewer, the authors comment that standard deviation was calculated and used to show variance in the data sets. However, the text reads "standard error" which is not standard deviation - as it is the standard deviation divided by the square root of the sample size. Please correct accurately and update in the text.Response: We apologize incorrect description. In the text, "standard error" was corrected to "standard deviation" (Line 130).Reviewer #3: This revised manuscript includes additional experiments examining the effects of IL-1ß and sclerostin on the expression of Wnt/ß-catenin signaling components at early and late stages of chondrogenic differentiation in ATDC5 cells. It appears that IL-1ß inhibits the expression of both positive and negative regulators of Wnt/ß-catenin signaling at early stages of chondrogenic differentiation, and promotes their expression in terminal calcification at late stages of chondrogenic differentiation. In addition, sclerostin counteracts the effects of IL-1ß at late stages but not at early stages. However, the present version failed to address the issues raised in my previous review. In particular, it remains unclear whether IL-1ß inhibits Wnt/ß-catenin signaling at early stages of chondrogenic differentiation, and promotes Wnt/ß-catenin signaling at late stages of chondrogenic differentiation.I understand that IL-1ß may exert differential effects at early and late stages of chondrogenic differentiation. Nevertheless, IL-1ß seems to regulate the expression of both positive and negative regulators of Wnt/ß-catenin signaling. Thus, the net outcome on Wnt/ß-catenin signaling is unclear.Response: IL-1β modulates the chondrogenic differentiation and Wnt/ß-catenin signaling, but the effects differ between the early and late stages. Previous studies reported that IL-1β impaired initial chondrogenic differentiation from mesenchymal cells . The mechanisms may be associated with downregulation of wnt/β-catenin signaling due to negative feedback by the interaction between Wnt/β-catenin signaling and the others such as NF-κB [3], although details are unclear. When the cells were treated with IL-1β from the beginning in the present in vitro culture system of chondrogenic differentiation, initial chondrogenic differentiation was inhibited and wnt/β-catenin signaling was downregulated in the early stage. But once chondrogenic differentiation was progressed, IL-1β upregulated wnt/β-catenin signaling in the late stage of chondrogenic differentiation and promoted endochondral ossification. This mechanism is supported by the other studies .References1. Simsa-Maziel S, Monsonego-Ornan E. Interleukin-1beta promotes proliferation and inhibits differentiation of chondrocytes through a mechanism involving down-regulation of FGFR-3 and p21. Endocrinology. 2012; 153: 2296-310.2. Murakami S, Lefebvre V, de Crombrugghe B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem. 2000; 275: 3687-92.3. Ma B, van Blitterswijk CA, Karperien M. A Wnt/β-catenin negative feedback loop inhibits interleukin-1-induced matrix metalloproteinase expression in human articular chondrocytes. Arthritis Rheum. 2012; 64:2589-2600.4. Mumme M, Scotti C, Papadimitropoulos A, Todorov A, Hoffmann W, Bocelli-Tyndall C, et al. Interleukin-1beta modulates endochondral ossification by human adult bone marrow stromal cells. Eur Cell Mater. 2012; 24: 224-36.5. Scotti C, Piccinini E, Takizawa H, Todorov A, Bourgine P, Papadimitropoulos A, et al. Engineering of a functional bone organ through endochondral ossification. Proc Natl Acad Sci U S A. 2013; 110: 3997-4002.The effect of sclerostin on IL-1ß at late stages of chondrogenic differentiation is likely indirect. Sclerostin binds to LRP5/6 to inhibit Wnt/ß-catenin signaling, and it is believed that the antagonistic effect of sclerostin on bone formation is mediated by Wnt signaling. Thus, there is no sufficient novelty in the present study.Response: IL-1β inhibits the expression of sclerostin/SOST in ATDC5 cells . As a possible mechanism, the stimulatory effects of IL-1β on Wnt/β-catenin signaling may be mediated by regulating interaction between sclerostin and LRP6. Suppression of sclerostin by IL-1β may upregulate LRP6 phosphorylation and downstream of Wnt/β-catenin signaling is thought to be activated. In this condition, addition of sclerostin canceled the upregulation of Wnt/β-catenin signaling.It is difficult to understand how the experiments were done, in particular, when the early effects were analyzed after treatment. For example, the results presented in Fig. 1C seem to be obtained after 3 weeks, but it is not clear when the cells were treated, and they represent early or late effect?Response: We apologize inaccurate description regarding treatment of cells. This point was revised in the methods section (Line 74). The cells were treated with IL-1β and/or sclerostin for 3 weeks as the early stage experiment. In the present in vitro culture system, it takes at least 7 weeks to complete whole process of chondrogenic differentiation from mesenchymal chondroprogenitor to terminal calcification. The chondrogenic differentiation initiates to form cartilage nodules through a cellular condensation process that gave rise to proliferating chondrocytes and no hypertrophic chondrocytes are present by 3 weeks. After 5 weeks, mineralized regions began to appear in the preexisting cartilage nodules as tiny spots and they propagated throughout the cartilage nodules with time. Thus, the cells at 3 weeks were included in the early stage.Attachmentresponse to reviewers_rev2.docxSubmitted filename: Click here for additional data file. 11 Sep 2020Sclerostin inhibits interleukin-1β-induced late stage chondrogenic differentiation through downregulation of Wnt/β-catenin signaling pathwayPONE-D-19-33198R2Dear Dr. Kumagai,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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For more information, please contact Kind regards,Michael SchubertAcademic EditorPLOS ONE 15 Sep 2020PONE-D-19-33198R2Sclerostin inhibits interleukin-1β-induced late stage chondrogenic differentiation through downregulation of Wnt/β-catenin signaling pathwayDear Dr. Kumagai:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. 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The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scaffolds GSK3β and CK1 to earmark β-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of β-catenin and oncogenesis. However, the molecular mechanism by which APC promotes β-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple β-catenin and Axin interacting sites, undergoes liquid–liquid phase separation (LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and β-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the β-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of β-catenin. APC functions downstream of the Wnt signalosome, and it is essential for the degradation of β-catenin in the absence of Wnt stimulation2. APC forms a complex, termed the “β-catenin destruction complex” or “Axin degradasome” composed of β-catenin, the scaffold protein Axin, and two kinases: GSK3β and casein kinase 1 (CK1)4. In the complex, proximity of β-catenin to the two kinases leads to β-catenin phosphorylation, which in turn facilitates its ubiquitination and proteosomal degradation. Both APC and Axin are key for the function of the β-catenin destruction complex3. Axin is able to interact with all components of the complex: β-catenin, GSK3β, CK1, APC and Axin itself, and is believed to be the major scaffold. APC harbors multiple interaction sites for β-catenin and Axin, and an oligomerization domain4. Although APC is absolutely essential for the assembly of the destruction complex5, it’s mechanistic role is not completely understood.APC mutations are present in ~ 80% of colorectal cancer cases7. Moreover, the punctate structures are membraneless and have high protein dynamics as determined by fluorescence recovery after photobleaching (FRAP) experiments8. Recent studies have revealed that many membraneless organelles are formed and maintained by LLPS11. This has led to a hypothesis that the punctate structures in Wnt signaling pathways are biomolecular condensates formed by LLPS12, an idea that is yet to be tested directly in vitro.The β-catenin destruction complex forms micrometer-scale punctate structures in cells15. APC has a long IDR that contains binding sites for β-catenin  and Axin 17  immediately downstream of the MCR, which is mostly disordered , probably due to the polymerization of the protein through its DIX domain21. Precipitation was found in multiple steps of the purification process (data not shown), resulting a low protein concentration in solution that prevent us from testing its LLPS ability. In contrast, we obtained enough APC 20R2-7 protein in buffers containing 300 mM NaCl or higher. Reducing NaCl concentration results in the formation of micrometer-sized spherical granules in the solution  experiments. SNAP-tagged 20R2-7 labeled with SNAP-Surface 549 fluorophore was spiked into the non-tagged 20R2-7 to aid in visualization of the protein. FRAP results showed that the fluorescence signal recovered within minutes after bleaching, indicating the movement of the molecules was dynamic within the droplets Fig. –2. Time The APC 20R2-7 consists of multiple functionally important elements, so we asked if any of them were essential for LLPS. We generated four additional recombinant APC fragments of shorter length than 20R2-7 Fig. . Protein22 in SW480 cells, a cell line derived from colorectal cancer sample that only expressed a truncated APC 1-1338 aa23. The fragments 20R2-3, 20R2-5 and 20R2-7 all localized in the cytoplasm 27. Overexpression of Axin forms punctate structures in cells in a concentration dependent manner28. We asked if elevated Axin protein concentration cooperates with APC IDR to promote LLPS in cells. To this end, we overexpressed human Axin1 together with APC IDRs in SW480 cells. Consistent with previous observations5, mCherry-Axin1 forms puncta in a fraction of cells 18 h after transfection. In cells co-transfected with mEGFP-APC 20R2-7, the Axin1 puncta became more prominent relative to the mEGFP control cells  are those with highest mCherry-Axin1 intensity in the population  consistent with previous observations that higher Axin1 concentration promotes puncta formation28. We noticed that an increasing number of cells that have medium mCherry-Axin1 intensity were also puncta positive in APC 20R2-7 co-expressing cells. The quantification results confirmed this observation  in cells. APC IDRs containing multiple SAMP motifs increased the valency of the complex, allowing Axin1 to form micrometer-scale biocondensates at a concentration where it wouldn’t form in the absence of APC IDR Fig. B,C.33. The biocondensates create physically separated compartments in the cytosol that is specifically enriched of the components of the complex. The high concentrated proteins and the dynamic nature within the compartment could allow phosphorylation and ubiquitination of β-catenin to be carried out efficiently. Moreover, the physically separated compartment ensures the specificity of enzymes such as kinases GSK3β and CK1 within the condensate. Its dynamic nature also allows rapid assembly and disassembly, which permits the precise temporal regulation by upstream signaling events.The biocondensate model of β-catenin destruction complex has been proposed recently3. Here we provide evidence supporting the model that the complex is formed by LLPS via multivalent interactions between the scaffold APC and Axin. We showed that multivalent APC IDRs are able to phase separate in vitro . It has been shown that some proteins drive phase transition through heavily negative charged amino acids and their coacervation with binding partnersE.coli expression, all fragments were inserted into pGEX6-P-1 vector . For fluorescence labeling of the proteins, a SNAP tag was added at the N-terminal of each fragment. For mammalian cell expression, mEGFP (monomer GFP with A206K mutation) or mEGFP-APC fragments were inserted into pLenti CMV Hygro DEST (w117-1) vector 40. Axin-1 full-length cDNA is N-terminal mCherry tagged and inserted into pLenti6.2/V5-DEST vector (Invitrogen). The C-terminal V5 tagged in the vector was disabled by adding a stop codon after Axin-1 cDNA. β-catenin WT or S45A mutant was cloned into pcDNA3.1 vector (Invitrogen) for mammalian expression. M50 Super 8 × TOPFlash and M51 Super 8 × FOPFlash were gifts from Randall Moon 41. pRL-SV40P was a gift from Ron Prywes 42.APC fragments cloned: 20R2-3 (1351–1552 aa); 20R2-5 (1351–1913 aa); 20R2-7 (1351–2070 aa); 20R4-7 (1553–2070 aa); 20R6-7 (1914–2070 aa). For 43, using the default long disorder prediction parameters.IDR prediction is done with IUPred2AE. coli BL21 strain at 16 °C for 20 h in the presence of 0.1 mM IPTG (Sigma), purified with glutathione sepharose  in protein lysis buffer . GST tag was removed by PreScission enzyme in cleavage buffer . The proteins were further purified by Size-exclusion chromatography on an AKTA-FPLC (Amersham BioSciences) equipped with a Superose 6 Increase 10/300 GL column . Fractions were checked with SDS-PAGE and coommassie blue staining. Fractions containing the protein were collected, concentrated to a final concentration of 100 µM, snap-froze in liquid nitrogen and store at -80 °C for future use. For SNAP-tagged proteins, the SNAP tag was labeled before storing with SNAP-Surface 549 nm (New England Biolabs) according to manufacturer’s manual.GST (Glutathione S-transferase)-tagged recombinant proteins (GST-APC fragments and GST-SNAP-APC fragments) were expressed in Proteins stored at − 80 °C were thawed on ice. An aliquot were taken to observe under a 60 × objective lens with an EVOS M5000 Imaging System (Thermo Fisher) using bright field with differential interference contrast (DIC). Only proceed when no aggregates or particles were found in the solution. To make proteins with different concentration in various NaCl concentrations, dilute the 100 uM stock protein with dilution buffer  in a PCR tube to achieve the desired protein  and NaCl concentration . The mixture was incubated at room temperature (around 25 °C) for 1 h before transferring to a µ-Slide 8 Well Glass Bottom Chamber (Ibidi) and observed with the same microscope and objective. Granules were determined by their signature Brownian movement in the solution before settling down to the bottom of the chamber.45 (version 1.50i) and intensity corrected and normalized as described below. To eliminate noise signal, background intensity was subtracted for all droplets in the frame. The intensity of each bleached area was then normalized to unbleached droplets in the same frame to remove the global photobleaching during image acquisition. The intensity of each bleached area was compared with the average intensity of pre-bleach (5 images) and plotted. The half time t1/2 was calculated based on a non-linear regression curve fit. Average t1/2 of 10 movies was used.Time lapse and FRAP experiments were carried out with a 3i spinning disk confocal microscope equipped with a 63 × objective lens (CSU-W1 SoRa). Protein APC 20R2-7 was prepared in the same way as salt diagram experiment, except that 5% of SNAP-549 nm labeled 20R2-7 was added to facilitate visualization. 20R2-7 protein concentration was 25 µM and NaCl concentration was 150 mM. The experiments were carried out between 30 and 60 min after making the sample. Image was taken every 5 (time lapse only) or 10 s (FRAP), 100 frames total. For FRAP experiment, each bleach area was equal or less than 50% of the total area of the selected droplet. FRAP images were quantified with NIH ImageJ software2.SW480 cells (ATCC) was cultured in Dulbecco’s modified Eagle’s Medium  supplemented with 10% fetal bovine serum and penicillin–streptomycin (Life Technologies) at 37 °C incubator with 5% COFor transfection, cells were plated on 0.1% gelatin (EMD Millipore) coated cover glasses  in a 24 well cell culture plate (Thermo Fisher). Transfection was done using lipofectamine 3000 reagent (Thermo Fisher) following manufacturer’s manual. Amount of plasmids used for each well of a 24 well plate: mEGFP or mEGFP-APC fragment, 150 ng; mCherry-Axin, 50 ng; FLAG-β-catenin, 150 ng.18 h after transfection, cells were fixed with 4% paraformaldehyde, washed and blocked with 2% BSA in PBS containing 0.3% Triton X-100 (PBST). Cells were then incubated with primary antibodies at 4 °C overnight, washed with PBS and incubated with secondary antibodies in PBST for 1 h at room temperature. Nuclear was stained with 0.5 µg/mL DAPI in PBST (Sigma) for 10 min and washed with PBS twice. The cover glasses were then removed from the plate and mounted on microscope slides (Fisher) with Fluoro-Gel (Electron Microscopy Sciences).Primary antibodies used: chicken-anti-GFP , rabbit-anti-RFP , mouse-anti-β-catenin , mouse-anti-FLAG M2 . Secondary antibodies used (1:500 dilution): donkey-anti-rabbit conjugated with Cy3 ; donkey-anti-chicken conjugated with Alexa Fluor 488 (Jackson ImmunoResearch #703-545-155); Goat-Anti-Mouse conjugated with Alexa Fluor 647 (Jackson ImmunoResearch 115-605-003).46. Due to limit of resolution, only puncta with a diameter equal or greater than 0.5 µm were identified and their size measured. mCherry-Axin intensity and β-catenin intensity were normalized to the endogenous β-catenin intensity in non-transfected cells (average intensity of over 1000 cells) on each slide before comparing across slides. Statistics and Plots were done with GraphPad Prism. The data did not pass a normal distribution test. Thus non-parametric one-way ANOVA  test and Dunn’s multiple comparisons with correction were used.Images of cells were taken on confocal microscopes, a Zeiss 780 confocal microscope or a 3i spinning disk confocal microscope (CSU-W1 SoRa). 40 × objective lens were used. For quantification, areas on each slide contain at least 150 transfected cells and 1000 non-transfected cells were imaged using the 3i spinning disk confocal microscope. Quantification of cell intensity and puncta size were done with CellProfiler (version 3.1.8)mCherry-Axin and/or mEGFP-APC fragment were cotransfected with luciferase reporter plasmids into SW480 cells. Three replicates were performed for each condition in a 96 well plate. Amount of plasmid used for each well: mCherry-Axin (10 ng); mEGFP-APC fragment (30 ng); M50 TOPFlash or M51 FOPFlash (20 ng); pRL-SV40P renilla luciferase reporter (20 ng) as a control for transfection. 18 h after transfection, cells were lysed, and firefly and renilla luciferase substrates added sequentailly according to the manual of Dual-Glo Luciferase Assay System (Promega). Luciferase emission was measured using a microplate reader . Firefly luciferase activity of each well was normalized to the renilla luciferase activity of the same well before comparing between different conditions. Three independent biological replicates were done.Cells were lysed with RIPA buffer and protein concentration determined by Bradford assay. The same amount of total proteins across samples was denatured by adding 5X SDS sample buffer and boiled at 100 °C for 15 min. 10 µg of total protein was loaded on a SDS-PAGE and blotted with anti-FLAG M2 (Sigma F1804) or anti-β-tubulin antibody (Sigma 05-661). Secondary antibody used was peroxidase conjugated goat-anti-mouse (Jackson Immunoresearch #715–035-151).Supplementary Information.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.


Long non-coding RNAs (lncRNAs) are pervasively transcribed in genome and emerging as a new player in tumorigenesis due to their functions in transcriptional, posttranscriptional and epigenetic mechanisms of gene regulation. As the most frequent malignancy and the foremost source of cancer mortality, lung cancer is a heterogeneous disorder. The most common type of lung cancer is Non-small cell lung cancer (NSCLC), occupying 85% of the total cases, and the main subtypes of NSCLC include lung adenocarcinoma (LAD), large cell carcinoma (LCC), and lung squamous cell carcinoma (LSCC). Recently, numerous lncRNAs have been reported to be strongly linked to NSCLC. In the present study, we found that a new lncRNA CBR3-AS1 is highly expressed in lung cancer. In addition, we also examined the expression of lncRNA CBR3-AS1 in 60 of LADs, 40 of LCCs and 40 of LSCCs patient samples, finding that CBR3-AS1 was specificity highly expressed in LAD cancer tissues. Mechanically, we discovered that CBR3-AS1 could regulate the proliferation, migration and invasion of LAD cells through targeting Wnt/β-catenin signaling.Real-time PCR, RNA-pulldown, RIP, western blotting, lentivirus transfection, luciferase reporter assays, cell proliferation assays, colony formation assays, wound healing scratch assays and transwell assays were employed to examine the relationship between lncRNA CBR3-AS1 and its regulation of Wnt/β-catenin signaling in LAD cells.LncRNA CBR3-AS1 is highly-expressed in LAD and cell lines. LncRNA CBR3-AS1 shows physical association with β-catenin. CBR3-AS1 could facilitate Wnt/β-catenin signaling activation thought promoting nuclear localization of β-catenin. CBR3-AS1 promotes LAD cell proliferation, migration and invasion by targeting Wnt/β-catenin signaling.It can be found that a new functional lncRNA CBR3-AS1 could promote nuclear localization of β-catenin so as to facilitate Wnt/β-catenin signaling activation and regulate the proliferation, migration and invasion of LAD cells. Lung cancer is considered as one of the most common cancers globally, affecting both genders . Two typRecently, high-throughput transcriptome analysis shows that nearly 75 percent of genome can be transcribed into RNAs and a few of them translate to proteins with others as noncoding RNAs (ncRNAs) , 9. LongIn recent years, an increasing number of evidences demonstrated that the lncRNAs exert important roles in a variety of diseases, especially in cancer. In NSCLC, there are a variety of lncRNAs with high expression levels as previous reports and some of them play significant roles. For example, CAR10 can bind and stabilize TFY-box-binding protein 1 (YBX-1) to up-regulate the expression of EGFR and proliferation of lung cancer cells . In addiWnt/β-catenin signaling pathway is an important molecular cascade in regulating embryonic development and adult homeostasis –23. Morehttp://www.rnanut.net/lncrnadisease/index.php/home/search), which was integrated comprehensive experimentally support and predicted ncRNA-disease associations curated from manual literatures and other resources [Long non-coding RNAs (lncRNAs) govern fundamental biochemical and cellular processes. To assess more important functional lncRNAs in non-small cell lung cancers (NSCLC), we examined the lncRNA-disease related database LncRNADisease v2.0  analysis, aiming to detect potential CBR3-AS1 associated proteins. This analysis identified β-catenin as a possible CBR3-AS1-interacting protein Fig. a. This iTo confirm the interaction between lncRNA CBR3-AS1 and β-catenin, we conducted another RNA pull-down assay and RIP assays in another LAD cell line, H1650 cells, indicating that the interaction between lncRNA CBR3-AS1 and β-catenin is prevalent in LAD cells Fig. d. In ordTo further characterize the function of CBR3-AS1, we transfected the control siRNA or CBR3-AS1 siRNA into A549 cells and also detected the expression level of β-catenin. The results suggested that knocking down CBR3-AS1 could not change the protein level of β-catenin . On this basis, numerous functional lncRNAs have been discovered in NSCLC. For example, HOTAIR can interact with polycomb repressive complex 2 (PRC2) and lysine-specific demethylase 1 (LSD1) to regulate some genes, thereby regulating the migration and invasion of NSCLC cells . The higIn recent years, a large number of studies on RNA-binding proteins have been published, and RNA-binding protein family developed enormously and confusedly. RNA-binding proteins (RBPs) belong to a class of proteins that bind to the double or single stranded RNA in cells and participate in the formation of ribonucleoprotein complexes. While RBPs can be combined with sequence and/or structural motifs in RNA via modular combinations of structurally identified RNA-binding domains (RBDs) , such asThe sources of antibodies against the following proteins were: β-actin  from Sigma; β-catenin ; Histone H3  from abcam.FLAG-β-catenin was carried by pLenti-Hygro vector. lncRNA CBR3-AS1 was carried by pcDNA3.1 vector. TopFlash DNA fragment coding eight tandem repeats of TCF response elements, and FopFlash DNA fragment as a negative control, carrying eight mutant TCF tandem repeats, were synthesized and introduced to pGL3-promoter vector upstream of the SV40 promoter to generate pGL3-TopFlash and pGL3-FopFlash respectively.A549, H23, H1650, H1975, H3122, HCC827, H1155 and 16HBE cells were got from the American Type Culture Collection  and cultured under the manufacturer’s instructions. All of the cultured cells were authenticated by examination of morphology and growth characteristics, and were confirmed to be mycoplasma-free.Cells were lysed by Laemmli sample buffer , and re-suspending in 5× SDS-PAGE loading buffer. The boiled protein samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane incubated with appropriately primary antibodies and secondary antibodies  in line with a Thermo Finnigan Surveyor MS Pump Plus HPLC system. The mass spectrometry analysis was carried out in a data-dependent mode with an automatic switch between a full MS and an MS/MS scan in the obitrap. Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Sequest was searched with a peptide tolerance of 3.0 Da and a fragment ion tolerance of 1.0 Da. The results of peptide sequences information of CBR3-AS1 interact proteins were offered in Additional file 7 cells were harvested and crosslinked with 0.3% formaldehyde for 10 min at RT and quenched with 0.125 M glycine for 5 min. Nuclei were extracted and lysed in RIP Cross-Linked Lysis Buffer and product protocol supplied by Magna Nuclear RIP™ (Cross-Linked) Nuclear RNA-Binding Protein Immunoprecipitation Kit . In addition, RNAs were extracted with TRIzol and detected by qRT-PCR.For A549 cells, 1 × 10In our studies, all siRNAs were transfect to cells using Lipofectamine RNAiMAX (Invitrogen) reagents following the manufacturer's recommendations. The final concentration of the siRNA molecules is 10 nM and cells were harvested 72 h. Control siRNA and the individual siRNAs against CBR3-AS1 were chemically synthesized by GenePharma. The short hairpin RNAs (shRNAs) against β-catenin or CBR3-AS1 were expressed as lentiviral, purchased from GenePharma, transfected into appropriate cells. The sequences of siRNAs and shRNAs are provided in Additional file Total cellular RNAs were isolated by TRIzol reagent (Invitrogen) and transcribed by the Reverse Transcription System (Roche). Quantitation of all gene transcripts was done by qPCR using a Power SYBR Green PCR Master Mix (Roche) and Q5 detection system (Thermo) with the expression of ACTB as the internal control. The primers used were listed in Additional file A549 cells stably expressing indicated genes or/and shRNAs were cultured in the absence or presence of LiCl for 2 weeks. After 2 weeks, the cells were washed and fixed with PBS and methyl alcohol respectively, then, stained with crystal violet (0.5% wt/vol). The number of colonies per well was counted.Indicated cells were cultured in appropriate media for 24 h in 96-well plates, 10 μl CCK-8 regents were added ahead of 2–4 h for test OD value of 450 nm. The detail protocols were supplied by Cell Counting Kit-8 .Experimental data from biological triplicate experiments are presented with error bar as mean ± SD. Two-tailed unpaired Student’s t-test was used for comparing two groups of data. Analysis of variance (ANOVA) with Bonferroni’s correction was used to compare multiple groups of data. A P value of less than 0.05 was considered significant and higher than 0.05 was considered no specific differences. All of the statistical testing results were determined by SPSS 22.0 software diagrams were conducted using GraphPad prism 8.0. Before statistical analysis, variation within each group of data and the assumptions of the tests were checked.Additional file 1: Figure S1. LncRNA CBR3-AS1 is highly-expressed in LAD and cell lines. (A) The expression of lncRNA CBR3-AS1 in LAD (n = 483) or Normal (n = 347) and LSCC (n = 486) or Normal (n = 338) tissues from TCGA and GTEx database (http://gepia.cancer-pku.cn/detail.php?gene=CBR3-AS1). **P < 0.01, #P > 0.05, one-way ANOVA. Figure S2. CBR3-AS1 facilitates Wnt/β-catenin signaling activation by promoting nuclear location of β-catenin in LAD cell lines. (A) A549 cells were transfected with control siRNA or CBR3-AS1 siRNA and the expression of c-Myc, LGR5 and MMP-7 was examined by western blotting assays. Figure S3. CBR3-AS1 promotes LAD Wnt/β-catenin signaling. (A) Quantitation colony number of colony formation assays with A549 cells stably expressing control shRNA or CBR3-AS1 shRNA and cultured in the absence or presence of 25 mM LiCl for 2 weeks. Each bar represents the mean ± SD for biological triplicate experiments. **P < 0.01, one-way ANOVA. (B) Growth viability assay with H1975 cells stably expressing control shRNA or CBR3-AS1 shRNA and cultured in the absence or presence of 25 mM LiCl for 8 days. Each bar represents the mean ± SD for biological triplicate experiments. 0.01 < *P < 0.05, **P < 0.01, one-way ANOVA. Figure S4. CBR3-AS1 regulates the LAD Wnt/β-catenin signaling. (A) Wound healing scratch assays with H1975 cells stably expressing control shRNA or CBR3-AS1 shRNA or co-transfected with FLAG-β-catenin. Each bar represents the mean ± SD for biological triplicate experiments. #P > 0.05, 0.01 < *P < 0.05, **P < 0.01, one-way ANOVA. Scale bar, 50 μm. (B) H1975 cells stably expressing control shRNA or CBR3-AS1 shRNA or co-transfected with FLAG-β-catenin followed by transwell invasion assays. Each bar represents the mean ± SD for biological triplicate experiments. #P > 0.05, **P < 0.01, one-way ANOVA. Scale bar, 50 μm.Additional file 2: Western blotting original data.Additional file 3: Table S2. Mass spectrometry analysis of lncRNA CBR3-AS1 interacting proteinAdditional file 4: Table S3. siRNA or shRNA sequence. Table S4. qRT-PCR primers.Additional file 5: Table S1. Patient information.


Many questions about how stem cells communicate with neighboring cells and self-organize to initiate tissue formation remain unanswered. We uncovered mechanisms employed by embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) to coform embryo-like structures. We describe ESC-generated cytonemes that react to self-renewal–promoting Wnt ligands secreted by TSCs. We identified glutamatergic activity upon formation of ESC–TSC interaction. This cellular connection is required for the transmission of Wnt signals to ESCs for Wnt/β-catenin pathway activation, a process that regulates morphogenesis. Given that many stem cell types express glutamate receptors and rely on niche-secreted Wnt ligands for self-renewal, we propose that Wnt and glutamatergic signaling crosstalk may prove prevalent in various mammalian tissues to regulate stem cell–niche interactions. Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC–TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal–promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC–TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC–TSC contact–mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification. Stem cells reside in cellular niches that impart chemical and physical signals to regulate their self-renewal and differentiation . UnderstWnt/β-catenin signaling has been implicated in tissue patterning and the self-renewal of many types of mammalian stem cells, including embryonic stem cells . This paDrosophila tissues and in other organisms, including Zebrafish  versus other Wnts that transduce β-catenin–independent pathways . Therefore, we immobilized purified Wnt3a and Wnt5a onto microbeads, distributed the microbeads around single ESCs, and investigated the interaction between cytonemes and Wnt beads. Our results indicate that ESCs can distinguish between signals and selectively reinforce a connection to the self-renewal Wnt3a ligand in an LRP6-dependent process. This signal recruitment is also mediated by the activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors at the cytonemes, which produces calcium transients. We identified the roles of intracellular calcium stores, Wnt receptors, DVL2, and β-catenin in regulating the formation and length of ESC cytonemes.In conclusion, we demonstrate that ESCs possess specialized cytonemes that react to self-renewal signals and orchestrate ESC–TSC pairing, setting the basis for spatial organization and specification of embryonic tissues.Movie S1). We did not observe TSCs contacting ESCs in a similar manner to establish ESC–TSC pairing.ESCs and TSCs possess the ability to self-sort and organize when cultured together to generate embryonic structures –4. By tiSI Appendix, Fig. S1A). Importantly, the interaction of ESCs and TSCs can result in activation of the Wnt/β-catenin pathway in ESCs, as indicated by the 7 oligomerized T cell factor (TCF)-binding sites (7xTCF)-eGFP . To verify if the TSCs were, in fact, the source of the Wnt ligands, we opted to use a short-term inhibition of Wnt ligand secretion to minimize the potential impact on TSC maintenance and identity. Accordingly, ESCs incubated with TSCs pretreated with inhibitor of Wnt production-2 (IWP2), a small molecule that blocks the secretion of Wnt ligands . These results indicate that TSCs produce Wnt ligands that are received by ESCs to activate the Wnt/β-catenin pathway.ESCs rely on activation of the Wnt/β-catenin pathway for self-renewal , 20. TheCF)-eGFP  ESC repo ligands , for 24 Movie S2). We obtained similar results using a different Wnt secretion inhibitor, Wnt-C59 . We speculated that the ESC protrusions are cytonemes that sense TSC-derived Wnt ligands, which are essential for the establishment of stable contacts during ESC–TSC pairing. To confirm this, we generated a double knock-out (dKO) of the Wnt coreceptors LRP5 and LRP6 in ESCs (LRP5/6dKO) and observed that the transient contact between cytonemes and TSCs was unaffected. However, these ESCs had a significantly reduced ability to establish stable contacts with TSCs, similarly to the ESC interaction with IWP2-pretreated TSCs . Our results suggest that specialized ESC cytonemes induce ESC–TSC pairing, an essential step in synthetic embryogenesis.Next, we determined whether the ESC–TSC interaction itself is affected by IWP2 treatment of TSCs. We observed that ESCs contact treated TSCs transiently via the protrusions; however, in 76% of cases this was not followed by ESC–TSC pairing  and monitored initial cell-bead contact by live imaging. Primary observations revealed that ESCs utilize thin cytonemes to contact the bead and can react by directing a larger cytoneme to recruit the bead to the plasma membrane (reactive interaction [RI]) to form a stable contact (Movie S3). Although Wnt5a has high protein sequence similarity to Wnt3a, our assay indicated a significantly higher proportion of reactive interactions when cytonemes encountered Wnt3a beads (76% RI) relative to Wnt5a beads (43% RI)  of single ESCs . Wnt5a, (43% RI) E. This sSI Appendix, Fig. S1D)—or to uncoated beads. Our results indicate that the cytonemes are unable to react to iWnt3a beads or uncoated beads efficiently  treated with dithiothreitol (DTT) to break the disulfide bridges in Wnt ligands, thus disrupting protein tertiary structure to render it inactive , although more cytonemes can form subsequently . Cytonemes are dynamic and retract upon cell rounding prior to division . These cytonemes are also present in ESC lines with different genetic backgrounds . The time required to generate cytonemes varies between different ESC lines, but after five hours the percentage of cells with cytonemes is similar . Scanning electron microscopy (SEM) revealed two major cytoneme types that both appear capable of interacting with the Wnt3a bead: thin cytonemes (∼100-nm length) emanating from the main body of the cell and other nanostructures that are generated from larger cytonemes  prevented the formation of new cytonemes and restricted the motility of both the cell and the existing cytonemes . Blockade of actin filament bundling using the Fascin inhibitor (Fascin-G2) also prevented cytoneme formation . Therefore, cells without bundled actin filaments lose the capacity to recruit beads. Conversely, inhibition of tubulin polymerization by Colcemid did not impact this process . Cytonemes contain components of the Wnt signaling pathway. On average, 58.9% of the observed cytonemes  contain LRP6 and all cytonemes have the downstream signaling component β-catenin . Contact with the Wnt source (Wnt3a bead or TSC) leads to the polarization of the Wnt coreceptor LRP6 and β-catenin in the ESC toward the area of contact  influx is often involved in neurotransmission and Ca2+ channels can be found on the pre- and postsynapse .Contact-mediated signaling shares similarities with the neuronal synapse . In bothtsynapse . We exam GCaMP6s  and empl GCaMP6s A and B. cytoneme A. Simila contact B and C.  contact B and C. 2+ from the media by ethylene glycol tetraacetic acid (EGTA) significantly reduced Ca2+ transients (only 25% of the cells) upon a stable contact between a Wnt3a bead and the cytoneme . These results suggest that Ca2+ influx is mediated by activated Ca2+ protein channels/transporters at the Wnt3a bead contact area. We sought to investigate those protein channels or transporters.Chelating Ca2+ transients at the cytonemes of Drosophila air sac primordium that are essential for Decapentaplegic (Dpp) signaling. These Ca2+ transients require glutamatergic activity mediated by the glutamate receptor GluRII. Crosstalk between ionotropic glutamate receptors, Ca2+ influx, and Wnt signaling has also been described in the neuronal synapse  and proteomic analysis indicate that ESCs express ionotropic glutamate receptors (iGluRs) (N-methyl-D-aspartate [NMDA] receptor) or cyanquixaline (CNQX), a competitive antagonist for the AMPA and kainate receptors. Only CNQX treatment significantly reduced the proportion of cells that produce Ca2+ transients at the cytonemes upon a stable contact with a Wnt3a bead (19%) . Consequently, CNQX treatment significantly reduced the proportion of reactive interactions with Wnt3a beads, thus impacting Wnt3a-bead recruitment to the plasma membrane . Importantly, the proportion of ESC cytonemes that establish a stable contact with TSCs and mediate ESC–TSC pairing, as well as the proportion of synthetic embryo structures .Huang et al.  recently synapse . We aime(iGluRs) , 31. Iniad (19%) C. Here, membrane D. The ag2+, and Ca2+ influx are required for cytoneme formation. CNQX and MK801 had no significant effect on cytoneme formation . Depleting intracellular Ca2+ stores by Thapsigargin   (a cell-permeable Ca2+ chelator) . However, Thapsigargin treatment did not affect the ability of the existing cytonemes to react to Wnt3a beads  , 33 or 1helator)  signific3a beads D. On the3a beads D. This s2+ on cytonemes that facilitate the recruitment of the self-renewal signal Wnt and mediate ESC–TSC pairing and the formation of synthetic embryos.In conclusion, the formation of ESC cytonemes is regulated by intracellular calcium stores. Activated AMPA/kainate receptors enable localized influxes of CaThe distribution patterns of Wnt receptors and downstream effectors such as β-catenin within cytonemes prior to cell-signal contact suggests that ESCs may utilize these proteins to regulate reactivity to an instructive signal. To examine this, we generated knock-out (KO) cell lines and studied the effect of the loss of each component on cytoneme formation and the recruitment of Wnt3a beads. Specifically, we generated LRP5KO, LRP6KO, LRP5/6dKO, and DVL2KO and utilized the published conditional β-catenin KO cell line (βKO) .SI Appendix, Supplementary Text and Figs. S6 and S7 for a full description of the generation and characterization of the KO cell lines.We initially characterized these cell lines including the expression of pluripotency markers, the protein levels of components of the Wnt/β-catenin pathway, and the ability of the different KO cell lines to activate the Wnt/β-catenin pathway. Please refer to SI Appendix, Fig. S6). Importantly, in this media CHIR 99021 (a glycogen synthase kinase 3 β [GSK-β] inhibitor) can activate the Wnt/β-catenin signaling pathway by inhibiting the destruction complex, overriding the need for Wnt proteins, the receptors, or DVL2 .Briefly, when cultured in 2i media that maintain ESC self-renewal , KO cell or DVL2 . The freIn the presence of purified Wnt3a ligands, leukemia inhibitory factor and serum-containing media LRP5KO has a significant reduction in activation of the Wnt/β-catenin pathway in comparison to WT, and LRP6KO is significantly further compromised. LRP5/6dKO cannot activate the Wnt/β-catenin pathway in this media. Furthermore, DVL2KO, which has elevated levels of DVL1 and DVL3, can activate the Wnt/β-catenin pathway even more than the WT cells in the presence of Wnt3a ligands A.Movie S4). Similarly, the cell lines LRP5KO, LRP6KO, and LRP5/6dKO have, on average, four cytonemes. LRP5KO cytonemes are marginally shorter when compared to WT cytonemes . On the other hand, LRP6KO has significantly shorter cytonemes (21.1 μm) than WT and the cytonemes are even shorter in the LRP5/6dKO cells (16.3 μm) . DVL2KO cells are rounder than WT cells and contain significantly fewer cytonemes (an average of three cytonemes) that are very short (10 μm) in comparison to all other studied cell lines . The round morphology is more prominent in βKO . The aforementioned statistically significant changes in the number and length of the protrusions of each KO cell line . Importantly, in these measurements the initial distance between the cell and the bead was similar for all cell lines .We next addressed whether the aforementioned differences between WT and mutant cells impact cytoneme formation and reactivity to niche signals. WT cells elongate and mainly produce bimodal cytonemes, but they can also have additional smaller cytonemes. Combined, these cytonemes, averaging five per single WT cell with an average maximum length of 29.9 μm, facilitate interaction with the surrounding Wnt3a beads . These results emphasize the essential crosstalk between Wnt coreceptors and Ca2+ transients at the cytonemes for the recruitment of localized Wnt signals and ESC–TSC pairing.All KO cell lines establish interactions with Wnt3a beads via cytonemes F. KnockiThe interactions of DVL2KO cytonemes are less reactive than those of the WT, whereas the reactivity of the βKO cytoneme interactions is unaffected G.Taken together, the evidence shows that LRP6 plays an important role in the initial recognition and subsequent recruitment of the Wnt3a bead to the plasma membrane after contact, while LRP5 is not required for this process. DVL2 and β-catenin regulate the length and number of cytonemes and the resulting efficiency of detecting the self-renewal signal without compromising its recruitment to the plasma membrane.2+ and promotes ESC–TSC pairing required for synthetic embryogenesis.In summary, we identified specialized ESC-produced cytonemes and the mechanism they use to detect and selectively react to self-renewal Wnts. This process requires crosstalk between Wnts and the family of glutamate receptors that involves an influx of CaDrosophila testis, thereby recruiting the ligand that is essential for their maintenance  receptors and extend to the hub cells in ntenance . IdentifHere, we show that ESCs use specialized cytonemes to distinguish between niche signals and preferentially select Wnt ligands promoting their self-renewal, such as those secreted by TSCs. After the recognition of the Wnt source, ESCs generate larger cytonemes to facilitate robust signaling and pairing with TSCs to promote synthetic embryogenesis.The multiplicity of secreted ligands and the difficulty of visualizing Wnt proteins in situ make it challenging to study how Wnt reception occurs in ESCs. To circumvent these issues, we utilized a reductionist approach of immobilizing purified Wnt ligands on microbeads, distributing them near single ESCs, and observing their interaction by time-lapse imaging.2+ transients. This crosstalk is required to allow the reactive interaction, Wnt signaling, and ESC–TSC pairing that form the basis of cellular communication and spatial self-organization.We found that ESCs generate actin-based cytonemes that contain Wnt receptors, which represent, on average, 60% of the total protrusions that ESCs form. After a Wnt source is detected, the bulk of the receptors and β-catenin polarizes toward the Wnt. A larger cytoneme enriched with Wnt receptors and AMPA/kainate receptor subunits form a stable contact with the Wnt source. Consequently, LRP6-AMPA/kainate receptor crosstalk is initiated and generates localized CaDrosophila air sac primordium (The finding that ESCs utilize iGluR-containing cytonemes indicates a striking similarity to aspects of the neuronal synapse. Both systems show selective, directed cellular protrusions for the purposes of reinforcing a “correct” signal and leading to higher-order spatial signaling and organization. Recent comprehensive investigations have demonstrated that components of the pre- and postsynapse are essential for cytoneme-mediated paracrine signaling in the imordium  and thatimordium . These f2+ stores and components of the Wnt pathway regulate cytoneme formation. Specifically, components of the Wnt pathway control the number of cytonemes, their length, and their selectivity to the ligand. LRP6, but not LRP5, regulates the length of the cytoneme. This may be attributed to motifs in the N terminal of LRP6 that can bind to regulators of actin filaments and promote their elongation, branching, and dynamics (Intracellular Cadynamics –41. Simidynamics . DVL2KO In summary, we identified specialized cytonemes that form the basis of ESC–TSC communication that is fundamental to morphogenesis. The minimal approach of single ESCs interacting with immobilized niche ligands provides unique insights into the mechanisms of contact-mediated signaling. We envision that mechanisms identified in this study could be applied to adult stem cells and niches where the Wnt/β-catenin pathway is required for self-renewal.SI Appendix.Details of the protocols for culture of ESCs and TSCs, generation and characterization of knockout ESC and reporter cell lines, purification and immobilization of Wnt proteins, conditions of cell imaging, image analysis, electron microscopy, flow cytometry, and statistical analyses performed can be found in SI Appendix as figures, additional legends, and references.All additional data and information are included in Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File