Irisin directly stimulates osteoclastogenesis and bone resorption in vitro and in vivo
Authors: Kenichi Nagano
Bruce M Spiegelman
Hyeonwoo Kim
Phuong T Le
Roland Baron
Eben G Estell
Mary L Bouxsein
Clifford J Rosen
Yosta Vegting
Christiane Wrann
Journal: eLife (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: bone; cell biology; irisin; mouse; myokine
Affiliations: Maine Medical Center Research Institute, Scarborough, United States, Dana Farber Cancer Institute, Boston, United States, Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States, Department of Medicine, Harvard Medical School, Boston, United States, Department of Cell Biology, Harvard University Medical School, Boston, United States, Beth Israel Deaconess Department of Orthopedic Surgery, Harvard Medical School, Boston, United States, Harvard School of Dental Medicine, Boston, United States

Abstract:  Irisin, a skeletal-muscle secreted myokine, facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In this study, we investigated whether irisin directly regulates osteoclasts. In vitro, irisin (2-10 ng/mL) increased osteoclast differentiation in C57BL/6J mouse bone marrow progenitors; however, this increase was blocked by a neutralizing antibody to integrin αVβ5. Irisin also increased bone resorption on several substrates in situ. RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating 'clastokines'. Forced expression of the irisin precursor Fndc5 in transgenic C57BL/6J mice resulted in lower bone mass at three ages and greater in vitro osteoclastogenesis from Fndc5-transgenic bone marrow progenitors. This study demonstrates that irisin acts directly on osteoclast progenitors to increase differentiation and promote bone resorption, supporting the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone. 

Retracted: No

PubPeer comments:
 Respected Authors, We have discussed your paper u201cIrisin directly stimulates osteoclastogenesis and bone resorption in vitro and in vivou201d with great interest and excitement during our weekly literature meeting. However, as we have noticed a few things that we would have liked to see explained: Can authors please update in which public repository RNA-seq data has been deposited. There is no information available in the published paper. Primer sequences used to generate figures 3b and 4a were not listed in the key resources table. This manuscript mentions a new mouse model that has Forced expression of Fndc5 in the murine muscle Mck promoter. No data related to generation or characterization of this transgenic mouse model including FNDC5 or irisin protein expression was shown. Only RNA expression data obtained interestingly from bone is shown in Figure 4a with a statement that irisin is highly expressed in bone marrow. *To confirm the capacity of irisin to impact osteoclast function in vivo, we employed a genetic strategy with chronically forced expression of Fndc5 using the muscle-specific Mck promoter. In that mouse model, we demonstrated that high levels of irisin were expressed in the whole bone marrow at both ages, and this was associated with low bone mass.* It is unclear from reading the paper how did authors arrive at this conclusion as no data was provided to support this conclusion. Can authors provide the evidence of forced expression of Fndc5 protein that was obtained as a part of characterization of this mouse model. Interestingly, looking in the summary of the peer-review file of this paper provided by the journal, it is mentioned that Fndc5 is an upstream regulator of irisin. *Using a combination of in vitro, RNAseq, and in vivo studies (transgenic mouse model over-expressing Fndc5, an upstream regulator of Irisin in response to exercise), the authors provide strong evidence that, in addition to its known effects on osteoblasts and osteocytes, irisin directly regulates osteoclasts.* This would be a wrong statement because Fndc5 is not an upstream regulator but a precursor of irisin peptide. As disclosed in the competing interest statement, the senior author is also a senior editor of the journal. , 
 Thanks for the note. We will provide the primer sequences. Happy to provide data on forced expression in other tissues: the RNA datA shows marked expression in several tissues besides muscle. We found 1500 fold higher mRNA expression in whole bone marrow at 8 wks of age and more than 500 fold higher expression at 13 months. Agree that the terminology for the Fndc5 precursor is confusing and could be stated differently. Will get back to you on deposition of the RNASeq data. Thanks for your comments , 
 #2 Thank you for your response. Leakiness of Mck promoter (if it is reproducible) in multiple tissues cell types apart from skeletal and cardiac muscle is indeed very concerning. Interestingly, There are publications that have used Mck promoter driven cre line for muscle specific expression making the data and interpretations that were reported in those papers possibly wrong. It would be very much appreciated if there are further details about the MCK-FNDC5 mouse line that was reported in the paper. As mentioned in the above comments, there are no details provided in the paper except to say that the line was obtained from some other lab. Details related to generation and characterization, in particular, about **Fndc5 protein levels** in this mouse model in muscle as well as in intact bone would be of interest and might be required to further support the basic conclusions mentioned in the paper. We look forward to primer sequences and RNA sequencing data that were not provided along with the published paper. Thank you and best wishes. , 
 Dear Dr. Rosen, It is stated in your article u201cOur previous work demonstrated that irisin concentrations in humans are approximately 2u20134 ng mL but increase to 10 ng mL or more with exercise, a dose range we employed for the in vitro studies (Jedrychowski et al., 2015).u201d In the article of Jedrychowski et al., irisin levels in plasma are shown to be slightly higher in aerobic interval trained subjects (4.33 ng mL) than sedentary controls (3.62 ng mL). I could not find data showing that irisin increases to 10 ng mL or more with exercise in that manuscript. I have not been able to find data showing an increase of circulating irisin to 10 ng mL upon exercise in any other manuscript either. I would be happy to know what manuscript you were referring to. , 
 #3 Primer sequences should have been downloaded by now from Dr Estell. If not shortly. We did not do protein measurements just m RNA And we agree with your conclusions about leakiness in other sites with other papers. Eric Olsen At UTSW made the Fndc5!transgenic With the Mck promoter and were provided to the spiegelman lab. . We donu2019t have those mice currently but dr Olsen might. Cliff Rosen , 
 #4 For the irisin in vivo analysis Lee et al Cell Metabolism shows the fold change in irisin after submaximal exercise , 
 Thank you for the answer. As far as I can see Lee et al identifies FNDC5 within a band that is 24 kDa after deglycosylation. Jedrychowski et al shows that irisin is located at 12 kDa. I don t think Lee et al has quantified the concentration to be 10 ng mL or more after exercise, and they might not have identified irsin but FNDC5. I therefore think that this sentence u201cOur previous work demonstrated that irisin concentrations in humans are approximately 2u20134 ng mL but increase to 10 ng mL or more with exercise, a dose range we employed for the in vitro studies (Jedrychowski et al., 2015).u201d is somewhat misleading (based on the two manuscripts you are referring to). , 
 I have a slide I can send you showing the increase in irisin with exercise; I thought it was in Lee but if not happy to share; , 
 ![file] here it is , 
 Dear Dr. Rosen, Yes, Lee et al show that a Western blot band located at 24 kDa is increasing after submaximal exercise (not significant with maximal exercise). They also identify a peptide fragment within the 24 kDa band that maps to FNDC5 or irisin with Mass spec. The size of the band they identify corresponds better with FNDC5 than irisin. Jedrychowski et al shows that irisin is located at 12 kDa. Irisin might increase by acute exercise, but I don t think it has been shown thoroughly yet (with methods used in the Jedrychowski paper). It would be interesting to show that irisin is in fact increasing to 10 ng mL or more with exercise in humans. Maybe your group or the Spiegelman group can test that? Have a good day. , 
 #1 Thank you for your interest in the manuscript and your comments. I have attached the primer sequences below (apologies for the image format only, limitation of PubPeer uploads). The full RNAseq results with fold change and statistical values has been submitted to the Dryad public repository per eLife s guidelines and linked to the original manuscript. As mentioned, we unfortunately do not have protein from this transgenic mouse which we had limited access to and is no longer in our hands, but as mentioned used mRNA in key tissues to confirm the increased expression of Fndc5, which would lead to higher levels of irisin via the normal cleavage process for its release. We are currently following up this work with other Cre-driven mouse models that will utilize other drivers in addition to McK to address the leakiness of this driver and seek to confirm the role of muscle-derived irisin in modulating bone cell activity with protein-level characterization. #4 Thank you for the comment, agreed that the wording and referencing of this sentence is misleading. The Jedrychowski citation was intended to refer only to the specific measurements in that paper to establish a more general log range of 1-10 ng mL as the physiologic levels (i.e. 10s not 100s ng mL). Interestingly other studies in human (Nygaard et al 2015 PLOS ONE, Daskalopoulou et al 2014, Eu J Endo) that rely on ELISA as opposed to mass spec have reported similar fold-increases with exercise (~1.5-2x), but at a higher range from baseline to post-exercise (150-450 ng mL). We began with dose studies in Fig. 1 to address such differences in reported levels, perhaps driven by differing measurement techniques, and as noted in the discussion found that even lower levels (similar to the mas spec measurements, 2 or 5 ng mL) increased osteoclast number, while levels in the 100s ng mL were inhibitory in our in vitro model. We thus selected 10 ng mL as an elevated dose for effective in vitro treatment to represent the high end of what we felt was the physiologic range, while still comparatively low to the ELISA measurements. This process of selecting 10 ng mL as the experimental parameter is similarly addressed in the results and discussion, but the reference in question to the Jedrychowski paper later in the discussion remains confusing, as we did not mean to imply that 10 ng mL came directly from the paper. The data Cliff provided above demonstrates precedence for a log-fold increase after exercise, which would support that 10 ng ml represents the top of a physiologic elevated range. **Primer Sequence List** *Sequences identified via PrimerBLAST (NCBI-NIH), procured from Integrated DNA Technologies (Coralville, IA)* ![file] , 
 #5 Thanks Dr. Rosen. If you can shed some light on what background strain used to generate the TG mice, copy number of the transgene and was the whole gene or coding region of human FNDC5 was used for transgenic mice generation. , 
 Thank you for your clarifications. I tend to be a little careful when I interpret the ELISA measurements on irisn. It has been reported a number of different irisin concentrations using ELISA kits (which might imply that the ELISA kits are unspecific). A meta-analysis from a couple of years back showed that the average increase in irisin (using ELISA) after acute exercise is 15% . So the increase after exercise should probably be ~15%, and not 2 fold or more, if you believe in the ELISA kit measurements... Anyway, I am probably a skeptical scientist, but I still donu2019t think that the true irisin concentration after acute exercise has been determined. And I think that most of the data so far argue for a relative modest increase in irisin after acute exercise. , 
 #13 I agree with you Helianthus Petiolaris. It is also interesting to note that background strain or any other information used to generate the MCK-FNDC5 mice was not indicated in the paper. Any differences in the background strain between control and MCK-FNDC5 mice will significantly affect the interpretations related to bone parameters. The strain differences will also explain the bone phenotypes or lack thereof between control and FNDC5 transgenic mice independent of irisin protein levels. , 
 Both the transgenic and their litter mates were on a C57BL6J background. The transgenic has been refreshed several times by backcrossing to B6 so that is not an issue For skeletal phenotypes , 
 #12 #12 #12 #12 Where can we find more data of FNDC5 transgenic mice used in this paper? There is no methodological data regarding this in the paper. , 
 Thanks for your inquiry; if you contact the third author of the paper, Christianne Wrann at MGH she will provide details thanks Clifford rosen , 
 #17 Thank you. Contacted but no response so far. Appreciate if you can contact him and get the details and shed some light on what background strain used to generate the TG mice, copy number of the transgene and was the whole gene or coding region of human FNDC5 was used for transgenic mice generation as this was not listed in you published manuscript. ,