Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3

Katelyn J. McCann; Manoj Yadav; Mohammadali E. Alishahedani; Alexandra F. Freeman; Ian A. Myles

doi:10.1371/journal.pone.0248011

Peer Review History

Original SubmissionJanuary 12, 2021
9 Feb 2021 Decision Letter - Pankaj K Singh, Editor PONE-D-21-01168 Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3 PLOS ONE Dear Dr. Myles, 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 provide additional data to support your conclusions, as suggested by the reviewers, or modify the text accordingly. Please submit your revised manuscript by Mar 26 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. 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. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Pankaj K Singh, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: Authors examine JAK2/STAT3 driven proliferation and metabolic dysregulation in keloid fibroblasts. Using JAK1/2 inhibitor ruxolitinib and 3 cell lines (healthy volunteer fibroblast cell line, keloid fibroblast cell line, and fibroblast cell line with STAT3 LOF) authors evaluated hyperproliferation and metabolic dysregulation by examining a) would closure; b) levels of OCR and ECAR; c) nuclear Hif1a; d) Tom20 levels; e) levels of mitochondrial pSTAT Ser727. Authors also examine whether addition of folic acid contributes to the altered proliferation and metabolic dysfunction in normal fibroblast cell line, keloid fibroblast cell line, and STAT3 LOF cell line. While it is an interesting study, experimental data does not necessarily directly support conclusions, and further experiments are required to validate the results. Major points: 1) Authors state that Warburg metabolism is driven by JAK2/STAT3 metabolic adaptation. While in some scenarios Warburg effect could be driven by JAK/STAT, most of the literature does not describe requirement for JAK/STAT pathway for Warburg effect. 2) Authors use STAT3 LOF cell line as a model of JAK/STAT hyperactive keloid cell line, however throughout the manuscript results from STAT3 LOF cell line are distinct from the Keloid cell line, suggesting that STAT3 LOF has a metabolic phenotype that does not match the one found in Keloid cell line. 3) What is the impact of reduced mitochondrial levels of pSTAT3 S727 in keloid cell line? The OCR levels in HV and Keloid cell lines are not significantly different (Figure 1C), suggesting that levels of pSTAT3 S727 (Figure 3E) are not directly contributing to differences in the OXPHOS. If so, what is the significance of pSTAT3 S727? 4) Conclusion that folic acid mediates effects on glycolysis are based on data that looks at indirect effects on glycolysis and mitochondrial ATP production across three cell lines. Current data does not directly support the hypothesis that folic acid mediates effects on glycolysis. Authors should show if inhibiting glucose uptake or glycolysis (for example by 2DG) prevents effects on cell lines treated with folic acid. Minor points: Line 47: Refs 5 and 10 show STAT3 plays a major role in the pathogenesis of keloids, but neither of the references mentions Warburg effect. Line 157: 2DG instead of 2GD. Reviewer #2: The manuscript entitled “Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3” by Myles et al submitted to PLOSOne described the potential role of JAK/STAT signaling and folate metabolism on the metabolic consequences during pathogenesis of keloid Scars. This is a well-designed study with a clear message and has a great clinical significance. I recommend publication of this article after addressing some of the minor comments. Comments: • Authors may explain the method how they isolated the fibroblast cells from health volunteer. • Figure 3 shown the immunofluorescence of HIF1a. This will be interesting if authors can validate this through western blotting of HIF1a protein. ****** 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. https://doi.org/10.1371/journal.pone.0248011.r001
Revision 1
11 Feb 2021 Author Response Reviewer #1: Authors examine JAK2/STAT3 driven proliferation and metabolic dysregulation in keloid fibroblasts. Using JAK1/2 inhibitor ruxolitinib and 3 cell lines (healthy volunteer fibroblast cell line, keloid fibroblast cell line, and fibroblast cell line with STAT3 LOF) authors evaluated hyperproliferation and metabolic dysregulation by examining a) would closure; b) levels of OCR and ECAR; c) nuclear Hif1a; d) Tom20 levels; e) levels of mitochondrial pSTAT Ser727. Authors also examine whether addition of folic acid contributes to the altered proliferation and metabolic dysfunction in normal fibroblast cell line, keloid fibroblast cell line, and STAT3 LOF cell line. While it is an interesting study, experimental data does not necessarily directly support conclusions, and further experiments are required to validate the results. Major points: 1) Authors state that Warburg metabolism is driven by JAK2/STAT3 metabolic adaptation. While in some scenarios Warburg effect could be driven by JAK/STAT, most of the literature does not describe requirement for JAK/STAT pathway for Warburg effect. We have amended the text throughout that JAK/STAT is an inducer of Warburg metabolism, but is not the lone pathway of importance (most notably in the introduction, lines 47-49). We continue to highlight that one of the described master regulators of Warburg metabolism (HIF1a) is highly linked to JAK/STAT signaling. We have also clarified in the text of the discussion that other signaling pathways may play a role (lines 293-294). 2) Authors use STAT3 LOF cell line as a model of JAK/STAT hyperactive keloid cell line, however throughout the manuscript results from STAT3 LOF cell line are distinct from the Keloid cell line, suggesting that STAT3 LOF has a metabolic phenotype that does not match the one found in Keloid cell line. We have clarified in the text that the STAT3 LOF cells are used to contrast against keloid cells – not to model them (lines 161-162). The keloid cells represent over-active STAT3 while the STAT3 LOF (loss of function) cells represent under-active STAT3. Thus, the cells lines’ distinctions are expected. 3) What is the impact of reduced mitochondrial levels of pSTAT3 S727 in keloid cell line? The OCR levels in HV and Keloid cell lines are not significantly different (Figure 1C), suggesting that levels of pSTAT3 S727 (Figure 3E) are not directly contributing to differences in the OXPHOS. If so, what is the significance of pSTAT3 S727? We have clarified in the text that the ultimate importance of pSTAT3Ser727 remains unclear (lines 256-257). We identified a baseline difference but that difference could not explain the entire phenotype. We opted against omitting our finding because (to our knowledge) it is the first direct evidence of a mito-STAT3 defect in keloid cells even if it does not explain keloid pathogenesis. Thus we acknowledge that we have more to elucidate as to the significance of our finding but the novelty of the finding is worthy of communicating to the scientific community. 4) Conclusion that folic acid mediates effects on glycolysis are based on data that looks at indirect effects on glycolysis and mitochondrial ATP production across three cell lines. Current data does not directly support the hypothesis that folic acid mediates effects on glycolysis. Authors should show if inhibiting glucose uptake or glycolysis (for example by 2DG) prevents effects on cell lines treated with folic acid. The experiment the reviewer is describing would assess if the effects of folic acid were mediated by glycolysis. However, we are asserting that the effects of folic acid are mediated by STAT3 and JAK1/2 – as indicated by the loss of folic acid effects in STAT3LOF cells and cells treated with JAK1/2 inhibition. We have attempted to clarify in the text. We have also cited publications that have previously established the effects of folic acid on induction of glycolysis (lines 216-217 and new references 36 and 37). Minor points: Line 47: Refs 5 and 10 show STAT3 plays a major role in the pathogenesis of keloids, but neither of the references mentions Warburg effect. We have amended the text to correct the descriptions. STAT3 in these papers is linked to keloid pathology, but separate work links STAT3 and Warburg physiology. We hope that our work now directly links the immunologic and metabolic pathways in STAT3. Line 157: 2DG instead of 2GD. This has been amended, thank you. Reviewer #2: The manuscript entitled “Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3” by Myles et al submitted to PLOSOne described the potential role of JAK/STAT signaling and folate metabolism on the metabolic consequences during pathogenesis of keloid Scars. This is a well-designed study with a clear message and has a great clinical significance. I recommend publication of this article after addressing some of the minor comments. We thank the review for their support, insights, and time. Comments: • Authors may explain the method how they isolated the fibroblast cells from health volunteer. We have clarified that the HV fibroblasts were also from cell lines purchased from ATCC. We opted to use a cell line to represent this group as it would improve reproducibility (other researchers can test these exact cells in their own lab). • Figure 3 shown the immunofluorescence of HIF1a. This will be interesting if authors can validate this through western blotting of HIF1a protein. We have attempted to clarify in the methods that since our findings are in the cells undergoing EMT/proliferation/migration – those cells are limited to the leading edge of the scratch. Western blotting would lack the ability to localize the expression of any protein and could only represent a lysate of the entire well. Furthermore, given that immunofluorescence is an established equal in its ability to perform protein quantitation as Western blotting, the need to assess only the cells in the scratched area indicates that the data as presented is the valid presentation. Attachments Attachment Submitted filename: Reviewer reply.docx https://doi.org/10.1371/journal.pone.0248011.r002
18 Feb 2021 Decision Letter - Pankaj K Singh, Editor Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3 PONE-D-21-01168R1 Dear Dr. Myles, 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, Pankaj K Singh, Ph.D. 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: Partly ****** 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: (No Response) Reviewer #2: (No Response) ****** 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.0248011.r003
Formally Accepted
22 Feb 2021 Acceptance Letter - Pankaj K Singh, Editor PONE-D-21-01168R1 Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3 Dear Dr. Myles: 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. Pankaj K Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248011.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .