Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver

Tomoko Nakata; Yuka Toyoshima; Takashi Yagi; Harumi Katsumata; Reiko Tokita; Shiro Minami

doi:10.1371/journal.pone.0235270

Peer Review History

Original SubmissionFebruary 7, 2020
13 Apr 2020 Decision Letter - Arun Rishi, Editor PONE-D-20-03557 Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver PLOS ONE Dear Dr. Nakata, 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. We would appreciate receiving your revised manuscript by May 28 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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 ****** 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: This group used subtraction hybridization to identify hepatic genes that are regulated by a single GH injection in hypophysectomized male rats. This study focuses on the GH-mediated regulation Rcan1 showing the Rcan1-4, but not the Rcan1-1, is upregulated by GH. Evidence is provided this is likely through the induction of Jun-c. The methods are solid, and for the most part the results are well described. The novelty of this work is based on the fact that we know very little regarding GH-mediated regulation of hepatic genes outside the vast literature focused on GHR-Jak2-Stat5b pathway. Comments and Concerns 1) It is assumed that the subtraction hybridization method revealed multiple transcripts that were differentially regulated. This information should be provided to the general scientific community. A rationale should be included as to why the regulation of Rcan was pursued in detail. It appears that this same group used the subtraction hybridization data to also investigate GH-mediated XBP-1 that has recently published in Endocr Journal 2020. 2) Ref 1 refers to the somatomedin (IGF1) hypothesis and would be best used after information supplied in line 41. Consider other reviews for the multiple actions of GH. 3) On line 56, when NFAT first appears, please define acronym. 4) At the end of the discussion, the last sentence is not linked to preceding information about the multiple GHR mediated intracellular signals. Perhaps include more information. "Since GH can activate multiple signal transduction pathways other than Stat5, we used subtraction hybridization to identify novel hepatic genes that may be regulated by these pathways, after a single injection of GH in hypophysectomized male rats. Rcan1 was identified and the GHR signal transduction pathway further explored. 5) human GH is used in the in vitro studies. Since hGH can also bind to the prolaction receptor, is there evidence that this receptor is not expressed by the H4IIE cells. Also for this cell line indicated methods of maintenance, passage number ... 6) Since some of the methods may have been already reported in detail in the recent Endocr J 2020 on GH mediated xBP1 expression, they could be shorten and reference to the paper cited. 7) In the results section lines 243/244 indicates Rcan1 "Which has not been report as a GH responsive genes" How was this determined? Give references as to the manuscripts and data bases searched. 8) Fig 3 and 4 are qPCR and expressed relative to Veh treated. For hypox rat expression what is the relative level of Rcan1-1 vs -4 expression with or without GH? Do you think it is expressed in hepatcytes, or other liver cell types. 9) Not clear why figure ligands are put in main text, it was difficult to follow. 10) The beginning sentence of the abstract, might suggest the study will be looking more at the role of Rcan in liver function instead of how Rcan is regulated by GH, might consider starting abstract differently. ****** 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 [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0235270.r001
Revision 1
26 May 2020 Author Response Response to Reviewers' Comments and Concerns 1) It is assumed that the subtraction hybridization method revealed multiple transcripts that were differentially regulated. This information should be provided to the general scientific community. A rationale should be included as to why the regulation of Rcan was pursued in detail. It appears that this same group used the subtraction hybridization data to also investigate GH-mediated XBP-1 that has recently published in Endocr Journal 2020. Reply After screening clones by differential hybridization, clones were further screened by virtual northern blot and positive clones were sequenced. And then they were analyzed by northern blot. Therefore we added method of virtual northern blot in Method (lines 118-130). “Clones that hybridized specifically to the forward probe were screened by virtual northern blot analysis. Virtual northern blots were blots of full-length cDNA, made by PCR amplification of the primary cDNA [29]. cDNAs were amplified with Advantage 2 Polymerase Mix. Cycle conditions were 95 ˚C for 1 min, then 17 cycles of 95 ˚C for 5 sec, 65 ˚C for 5 sec, and 68 ˚C for 6 min in a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA). The PCR product was separated on a 1.2% agarose gel, denatured and subsequently blotted onto a nylon membrane in a conventional southern transfer and UV-crosslinked. Each clone was labeled with PCR DIG Probe Synthesis Kit (Roche). Prehybridization, hybridization, washing and detection were performed according to the manufacturers' protocols. Clones that showed differential expression were sequenced by the BigDye Terminator Cycle Sequencing method using a 377 ABI Prism automated DNA sequencer (PerkinElmer) according to the manufacturers' protocols.” We added data in S1Table and Fig 1A, and described a rational as follows (lines 216-229). “From the forward subtracted library (subtracting vehicle-treated cDNAs from GH-treated cDNAs), 702 colonies were isolated. 48 clones were hybridized with only forward subtracted probe but not reverse subtractive probe (subtracting GH-treated cDNAs from vehicle-treated cDNAs). To identify the clones that were really upregulated by GH, they were analyzed by virtual northern blot and 8 clones that were upregulated by GH were sequenced (S1 Table). mRNA levels of these genes were further determined by northern blotting to confirm that GH regulated the expression of these genes. We found that the levels of Rcan1, Xbp1, Nampt, and Leap2 mRNAs were much higher at 1 h after GH injection than those after vehicle injection (Fig 1A). These genes have not been described as GH responsive genes [31], although Wauthier and Waxman indicated in microarray data that Rcan1 and Nampt mRNAs were increased by GH [32]. We have studied Rcan1 and Xbp1 in detail because GH increased the levels of RCAN1 and XBP1 proteins. We reported about Xbp1 previously [28].” 2) Ref 1 refers to the somatomedin (IGF1) hypothesis and would be best used after information supplied in line 41. Consider other reviews for the multiple actions of GH. Reply Ref 1 was moved to line 41 and replaced by the following review papers in line 38. 1. Vijayakumar A, Novosyadlyy R, Wu Y, Yakar S, LeRoith D. Biological effects of growth hormone on carbohydrate and lipid metabolism. Growth Horm IGF Res. 2010;20(1):1-7. 2. List EO, Sackmann-Sala L, Berryman DE, Funk K, Kelder B, Gosney ES, et al. Endocrine parameters and phenotypes of the growth hormone receptor gene disrupted (GHR-/-) mouse. Endocr Rev. 2011;32(3):356-86. 3) On line 56, when NFAT first appears, please define acronym. Reply NFAT first appears on line 53 and we defined acronym (lines 52-53). “Calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT)” 4) At the end of the discussion, the last sentence is not linked to preceding information about the multiple GHR mediated intracellular signals. Perhaps include more information. "Since GH can activate multiple signal transduction pathways other than Stat5, we used subtraction hybridization to identify novel hepatic genes that may be regulated by these pathways, after a single injection of GH in hypophysectomized male rats. Rcan1 was identified and the GHR signal transduction pathway further explored. Reply Thank you for the suggestion. According to your suggestion, we added the following sentences (lines 498-501). “In conclusion, since GH can activate multiple signal transduction pathways other than Stat5, we used subtraction hybridization to identify novel hepatic genes that may be regulated by these pathways, after a single injection of GH in HPX male rats. Rcan1 was identified and the GHR signal transduction pathway further explored.” 5) human GH is used in the in vitro studies. Since hGH can also bind to the prolaction receptor, is there evidence that this receptor is not expressed by the H4IIE cells. Also for this cell line indicated methods of maintenance, passage number ... Reply Yes, human GH has lactogenic effects through binding to PRL receptor. Exclude the possibility, we studied the effect of PRL on the expression of Rcan1-4 in vivo, and the result was PRL did not increase Rcan1-4 mRNA significantly. We added the data in Fig 2D, and the following sentences (lines 259-261). “Injection of the same amount of PRL did not significantly increase Rcan1-4 mRNA, which indicated that GH but not PRL increased Rcan1-4 mRNA (Fig 2D).” We also added the following sentences in Methods (lines 149-152). “H4IIE cells expressing the Ghr mRNA (H4IIE GHR) were constructed previously [28], and stored in alpha modified Eagle’s medium containing 10% fetal calf serum and 10% DMSO in liquid nitrogen freezer. H4IIE GHR cells at 16-20 passages were used in experiments.” 6) Since some of the methods may have been already reported in detail in the recent Endocr J 2020 on GH mediated xBP1 expression, they could be shorten and reference to the paper cited. Reply We shorten the methods. 7) In the results section lines 243/244 indicates Rcan1 "Which has not been report as a GH responsive genes" How was this determined? Give references as to the manuscripts and data bases searched. Reply Rcan1, Xbp1, Nampt, and Leap2 mRNAs have not been described as GH responsive genes in the paper of Flores-Morales et al (Flores-Morales A, Ståhlberg N, Tollet-Egnell P, Lundeberg J, Malek RL, Quackenbush J, et al. Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat. Endocrinology. 2001;142(7):3163-76.). Wauthier and Waxman provided the microarray data in the supplementary data on the web site of the paper (Wauthier V, Waxman DJ. Sex-specific early growth hormone response genes in rat liver. Mol Endocrinol. 2008;22(8):1962-74.), which indicated that Rcan1 and Nampt were GH responsive genes. Therefore we added their papers as the references and the following sentence (lines 223-229). “We found that the levels of Rcan1, Xbp1, Nampt, and Leap2 mRNAs were much higher at 1 h after GH injection than those after vehicle injection (Fig 1A). These genes have not been described as GH responsive genes [31], although Wauthier and Waxman indicated in microarray data that Rcan1 and Nampt mRNAs were increased by GH [32]. We have studied Rcan1 and Xbp1 in detail because GH increased the levels of RCAN1 and XBP1 proteins. We reported about Xbp1 previously [28].” 8) Fig 3 and 4 are qPCR and expressed relative to Veh treated. For hypox rat expression what is the relative level of Rcan1-1 vs -4 expression with or without GH? Do you think it is expressed in hepatcytes, or other liver cell types. Reply We added these sentences in Discussion (lines 412-424). “GH increased the 3.0-kb and 2.0-kb Rcan1 mRNAs in rat liver. The increased mRNAs were both likely to be Rcan1-4 mRNAs because GH did not increase Rcan1-1 mRNA, which indicated that the levels of Rcan1-4 mRNA were more abundant than those of Rcan1-1 mRNA when GH existed. We also found that RCAN1-4 protein level in HPX rat liver was less than that of normal rat liver in both male and female rats. This suggests that GH regulates RCAN1-4 protein levels in normal rat liver. Anti-RCAN1 antibody detected 40 kDa protein, which corresponds to RCAN1-1 protein suggesting that small amount of RCAN1-1 protein existed in the HPX liver. RCAN1-1 protein levels were not changed by injection of GH as Rcan1-1 mRNA were. Therefore RCAN1-4 protein seemed to be more abundant than RCAN1-1 protein when GH existed and almost equally scarce when GH did not exist. Because Rcan1-4 mRNA is expressed in H4IIE hepatoma cells, it is expressed in hepatocyte, although it may be expressed in other liver cell types in the liver.” 9) Not clear why figure ligands are put in main text, it was difficult to follow. Reply We put the figure ligands in main text because of PLOS ONE's style requirements. “Each figure caption should appear directly after the paragraph in which they are first cited.” http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf 10) The beginning sentence of the abstract, might suggest the study will be looking more at the role of Rcan in liver function instead of how Rcan is regulated by GH, might consider starting abstract differently. Reply We changed the beginning of the abstract as follows (lines17-20). “Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0235270.r002
12 Jun 2020 Decision Letter - Arun Rishi, Editor Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver PONE-D-20-03557R1 Dear Dr. Nakata, 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, Arun Rishi, 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: The authors have addressed all prior concerns and there are no additional concerns. ................. ****** 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 https://doi.org/10.1371/journal.pone.0235270.r003
Formally Accepted
18 Jun 2020 Acceptance Letter - Arun Rishi, Editor PONE-D-20-03557R1 Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver Dear Dr. Nakata: 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 Prof Arun Rishi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235270.r004

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