Phlpp1 is induced by estrogen in osteoclasts and its loss in Ctsk-expressing cells does not protect against ovariectomy-induced bone loss

Marcelline K. Hanson; Ismael Y. Karkache; David H. H. Molstad; Andrew A. Norton; Kim C. Mansky; Elizabeth W. Bradley

doi:10.1371/journal.pone.0251732

Peer Review History

Original SubmissionNovember 17, 2020
11 Feb 2021 Decision Letter - Jung-Eun Kim, Editor PONE-D-20-36058 PHLPP1 is induced by Estrogen in osteoclasts and its loss in Ctsk-Expressing Cells Does Not Protect Against Ovariectomy-Induced Bone Loss PLOS ONE Dear Dr. Bradley, 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. Please submit your revised manuscript by Mar 14 2021 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 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. 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Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The 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. Reviewer #1: Yes Reviewer #2: 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: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please 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. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the manuscript “Phlpp1 is induced by Estrogen in osteoclasts and its loss in Ctsk-Expressing Cells Does Not Protect Against Ovariectomy-Induced Bone Loss” Hassan MK et al have described the influence of Phlpp1 in osteoclast activity in overiectomy mouse model in presence or absence of estrogen. The work might have a good contribution in osteoclast biology. However, the following issues need to be addressed for its improvement 1. Authors might explain about the “Ctsk-Cre expressing cells”, and full form of CTSK 2. Full form of IGFBP4 3. Author should explain the procedure of isolation of osteoclast progenitor cells and its purity. 4. In figure 5, authors may show representative photos of TRAP positive osteoclast cells. 5. In figure 1, it would be nice if authors can show few photos of scanned via micro-CT 6. In figure 4, authors may provide the densitometry data of western blotting. Reviewer #2: In this manuscript, Hanson et al. document the effect of Ctsk-conditional Phlpp1 deletion on OVX-bone loss, and identify a potential mechanism by which estrogen regulates Phlpp1 and Igfbp4 expression. The underlying mechanism is very intriguing, however, there is a major issue with the overall premise. Specifically, since estrogen has effects on other bone cell lineages, including the osteoblast/osteocyte lineage, a loss of the increased bone phenotype with OVX does not specify a predominant role of Phlpp1 in resorption as compared to bone formation. In addition, there was no assessment of bone formation parameters in this model, beyond BV/TV, to support a lack of effect on coupling/bone formation. There are additional in vitro experiments/data that would strongly benefit this study. Major comments: 1. As stated above, the rationale behind using OVX to test specificity for a resorptive vs formation effect of the Phlpp1 Ctsk-cre KO is flawed, as estrogen has effects on the osteoblast/osteocyte lineage, and OVX is known to impact osteoblast differentiation/bone formation. 2. The authors show there is no significant difference in bone phenotype between OVX WT and Ctsk Cre Phlpp1 KO animals. However, given that the sham phenotype is different, the authors should also assess percent change compared to sham. The fact that the animals have the same bone phenotype following OVX would suggest that the there was a more negative effect of OVX on bone mass in KO as compared to WT animals. 3. The authors showed that E2 increase Phlpp1 and decrease Igfbp4 protein levels and that Phlpp1 KO osteoclasts exhibited increased Igfbp4 expression. In order to fully understand this relationship and the impact on bone phenotype, the authors should do the same time course E2 treatment on WT and Phlpp1 KO osteoclasts. 4. In order to assess the impact of Ctsk Cre Phlpp1 KO or OVX on bone formation, the authors should assess dynamic bone formation rates and osteoblast number by histomorphometry. Current data only are reflective of the osteoclast data. This study would also benefit from serum markers of resorption and formation. 5. Given that osteoclasts could secrete IGFBP4, could this be a potential E2 induced coupling factor that increases bone formation in the sham mice? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Reviewer comment-PLOS ONE.pdf https://doi.org/10.1371/journal.pone.0251732.r001
Revision 1
12 Apr 2021 Author Response Reviewer #1: In the manuscript “Phlpp1 is induced by Estrogen in osteoclasts and its loss in Ctsk-Expressing Cells Does Not Protect Against Ovariectomy-Induced Bone Loss” Hassan MK et al have described the influence of Phlpp1 in osteoclast activity in overiectomy mouse model in presence or absence of estrogen. The work might have a good contribution in osteoclast biology. However, the following issues need to be addressed for its improvement 1. Authors might explain about the “Ctsk-Cre expressing cells”, and full form of CTSK We delineated the Ctsk abbreviation within the manuscript; Ctsk-Cre expressing cells is used because the Ctsk-Cre transgene is expressed by different cell types. 2. Full form of IGFBP4 We defined Igfbp4 within the manuscript. 3. Author should explain the procedure of isolation of osteoclast progenitor cells and its purity. We added this detail to the methods section of our manuscript. 4. In figure 5, authors may show representative photos of TRAP positive osteoclast cells. We added these images to Figure 5. 5. In figure 1, it would be nice if authors can show few photos of scanned via micro-CT Unfortunately, we do not have current access to some of the scans needed to generate reconstructions. Reviewer #2: In this manuscript, Hanson et al. document the effect of Ctsk-conditional Phlpp1 deletion on OVX-bone loss, and identify a potential mechanism by which estrogen regulates Phlpp1 and Igfbp4 expression. The underlying mechanism is very intriguing, however, there is a major issue with the overall premise. Specifically, since estrogen has effects on other bone cell lineages, including the osteoblast/osteocyte lineage, a loss of the increased bone phenotype with OVX does not specify a predominant role of Phlpp1 in resorption as compared to bone formation. In addition, there was no assessment of bone formation parameters in this model, beyond BV/TV, to support a lack of effect on coupling/bone formation. There are additional in vitro experiments/data that would strongly benefit this study. Major comments: 1. As stated above, the rationale behind using OVX to test specificity for a resorptive vs formation effect of the Phlpp1 Ctsk-cre KO is flawed, as estrogen has effects on the osteoblast/osteocyte lineage, and OVX is known to impact osteoblast differentiation/bone formation. We revised our manuscript to reflect the Reviewer’s comments and added discussion of this limitation to our revised manuscript. 2. The authors show there is no significant difference in bone phenotype between OVX WT and Ctsk Cre Phlpp1 KO animals. However, given that the sham phenotype is different, the authors should also assess percent change compared to sham. The fact that the animals have the same bone phenotype following OVX would suggest that the there was a more negative effect of OVX on bone mass in KO as compared to WT animals. We added these comparisons to Table 1 and discussed them within our revised manuscript. 3. The authors showed that E2 increase Phlpp1 and decrease Igfbp4 protein levels and that Phlpp1 KO osteoclasts exhibited increased Igfbp4 expression. In order to fully understand this relationship and the impact on bone phenotype, the authors should do the same time course E2 treatment on WT and Phlpp1 KO osteoclasts. We added these data to Figure 4 and discussed them within our revised manuscript. 4. In order to assess the impact of Ctsk Cre Phlpp1 KO or OVX on bone formation, the authors should assess dynamic bone formation rates and osteoblast number by histomorphometry. Current data only are reflective of the osteoclast data. This study would also benefit from serum markers of resorption and formation. We added these data to our manuscript. 5. Given that osteoclasts could secrete IGFBP4, could this be a potential E2 induced coupling factor that increases bone formation in the sham mice? We added this provocative idea to our discussion. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0251732.r002
3 May 2021 Decision Letter - Jung-Eun Kim, Editor PHLPP1 is Induced by Estrogen in Osteoclasts and its Loss in Ctsk-Expressing Cells Does Not Protect Against Ovariectomy-Induced Bone Loss PONE-D-20-36058R1 Dear Dr. Bradley, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at 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. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jung-Eun Kim Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 addressed Reviewer #2: All comments have been addressed ******** 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The 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. Reviewer #1: Yes Reviewer #2: 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: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please 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. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Authors have addressed queries raised by reviewer. This revision has improved the quality of the manuscript. Reviewer #2: The authors addressed all of my comments. However, I don't see the edited figures in the resubmission. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0251732.r003
Formally Accepted
8 Jun 2021 Acceptance Letter - Jung-Eun Kim, Editor PONE-D-20-36058R1 Phlpp1 is Induced by Estrogen in Osteoclasts and its Loss in Ctsk-Expressing Cells Does Not Protect Against Ovariectomy-Induced Bone Loss Dear Dr. Bradley: 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. 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. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Jung-Eun Kim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0251732.r004

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