Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1
Authors: Mayuri Khandelwal
Rajiv Ranjan Singh
Viji Vijayan
Sarika Gupta
Kapil Manglani
Avadhesha Surolia
Journal: Free Radical Biology and Medicine (2013)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Forkhead box; Free radicals; Osteoprotegerin; Protein phosphatase 2A; Receptor activator of nuclear factor-κB ligand
Affiliations: Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India., Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Electronic address: ., Molecular Sciences Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India; Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560 012, India. Electronic address: .

Abstract:  In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1β, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality. 

Retracted: No

PubPeer comments:
 The images are duplicated in the same publication. Could the authors calrify. ![file] , 
 The same immunoblots with different treatment conditions The same immunoblot for b-actin and t-JNK Could the authors clarify this too? ![file] , 
 #1 Thank you for pointing out the similarities in images in Figure 1D (i), Figure 2B (iv) and Figure 3H(i). All the three images represent FOXO1 at basal conditions. These figures are mentioned as representative images. The authors add that another image Figures 2Bi is also FOXO1 under basal condition. The objective is to demonstrate that no FOXO1 nuclear localisation is seen under basal conditions as per cell culture experiments done in the study. These images actually had to be one figure but split to make findings clear to reader. Its a story split into many parts. Once again thank you for pointing this out. The authors will address this issue. , 
 #2 The authors are very thankful for pointing out this error. Figure 3D represents JNK blot and this is mentioned in the figure legend and results section. This blot that is under question is that of JNK and not beta-actin Figure 4C is not of beta-actin and this addition into Figure 4C is an error during figure presentation. This has occurred by mistake. We will write to the editor of Free Radical Biology and Medicine (FRMB) pointing this and will do needful to remove the blot from Figure 4C. Any other queries from the Editor, FRBM will be addressed in this regard. Once again thank you for pointing this out. Further updates on this will be posted in this platform. , 
 #3 Thank you for the response. The image orientation of the Figure 3H (i) is different to that of the other similar images in the publication; that is far from the author response, and still questions the genuinity of experimental data. In addition, the authors did not mention about the same images were reused in the all experimental conditions in any part of the publication. Besides, in such same conditions with the same groups, the authors could have provided seperate figures as then it would be believed to come from different independent experiments. In in vitro cultures, as each experiments to be conducted simultaneously and independently at the same conditions (especially in the experiments with treatment conditions) would provide a legible results. If one part of the experiment was conducted 2 days before and other started today may change the experimental system faulty and yield false positive results. Critically, at this moment, could the authors provide the raw data for their problematic figures including Figure 1D (i), Figure 2B (iv) and Figure 3H(i) from the other 2 independent experiments conducted to convince the audience. It would be appreciable if the raw data would be presented in a same fashion as shown in Figure 1D (i), Figure 2B (iv) and Figure 3H(i). , 
 This is another suspected critical image duplication. The immunoblot bands for t-AKT and PP2A are exactly the same. Authors are kindly requested to provide the original uncropped image, which is in the full membrane exposed picture captured. Also show the molecular weight marker in the same. The band highlighted in green seems to be a fabricated band, as i can clearly see some changes in my image editing software. The PP2A band is intact, though the lane 6 of p-PP2A is added seperately. Could the authors provide the original uncropped image, which is in the full membrane exposed picture captured. Also show the molecular weight marker in the same. ![file] , 
 In Figure 3 I; there is a bunch of discrepancies noted in the immunoblots. The experimental data presented would impact the conclusion of the paper and the authors should address a retraction. ![file] , 
 Could the authors clarify this duplication in the images? ![file] , 
 The immunoblots were exactly the same in another publication, mentioning a different treatment condition. Could the authors clarify this? Could the authors provide the original uncropped image, which is in the full membrane exposed picture captured. Also show the molecular weight marker in the same. ![file] , 
 For the experiments conducted with High Met + Ovx (Fig.5, 6,7 and 8), the dosage of Met was not mentioned in the figure figure legends or in the text (in material and methods). Could the authors explain the dosages used? , 
 Thanks for pointing out we will do the needful. , 
 The fold times in the two publications are different in the same conditions (same authors). Could the authors please clarify? ![file] ,