Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by nuclear factor-kappa B activation in osteoblastic cells
Authors: Carlos Guillén
Arancha R de Gortázar
María Eugenia Martínez
Pedro Esbrit
Pilar Martínez
Journal: Journal of Biological Chemistry (2002)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Parathyroid hormone; PTHrP; bone resorption; interleukin-6; IL-6
Affiliations: Bone and Mineral Metabolism Laboratory, Research Unit, Fundación Jiménez Díaz, 28040 Madrid, Spain.

Abstract:  Parathyroid hormone (PTH)-related protein (PTHrP) seems to affect bone resorption by interaction with bone cytokines, among them interleukin-6 (IL-6). Recent studies suggest that nuclear factor (NF)-kappaB activation has an important role in bone resorption. We assessed whether the N-terminal fragment of PTHrP, and its C-terminal region, unrelated to PTH, can activate NF-kappaB, and its relationship with IL-6 gene induction in different rat and human osteoblastic cell preparations. Here we present molecular data demonstrating that both PTHrP (1-36) and PTHrP (107-139) activate NF-kappaB, leading to an increase in IL-6 mRNA, in these cells. Using anti-p65 and anti-p50 antibodies, we detected the presence of both proteins in the activated NF-kappaB complex. This effect induced by either the N- or C-terminal PTHrP domain in osteoblastic cells appears to occur by different intracellular mechanisms, involving protein kinase A or intracellular Ca(2+)/protein kinase C activation, respectively. However, the effect of each peptide alone did not increase further when added together. Our findings lend support to the hypothesis that the C-terminal domain of PTHrP, in a manner similar to its N-terminal fragment, might stimulate bone resorption. These studies also provide further insights into the putative role of PTHrP as a modulator of bone remodeling. 

Retracted: No

PubPeer comments:
 The secondary land of IL-6 (Red box) was cut and spliced in to a RT-PCR picture. ![file] , 
 Figure 1: More similar than expected. Could authors check provide a higher quality image, please? ![file] Figure 2B: More similar than expected. Could authors check please? ![file] , 
 Figure 12: More similar than expected. Could authors check please? ![file] , 
 Figure 13: Contrast enhanced, more similar than expected. Could authors check please? ![file] , 
 #2 It s quite clear that also first and last lanes are duplicates (blue boxes). Could the authors please comment? ![file] , 
 Thanks to Charaxes nandina, Austroasca histrionicula and Aneurus inconstans for your comments. This paper was published 20 years ago and we cannot access to the raw data of the experiments because the laboratory was disassembled several years ago and it is impossible to localize any of the original experiments made in this lab. In addition to that, the PI retired several years ago. I would like to have the original scanned images in order to discuss with all of you and determine what could have happened with the images indicated in the comments. I should say that this paper is a big one (for that time), with many figures (13 figures) with different cell lines (UMR106, MG63 and human osteoblastic cells). All the experiments were repeated at least 3-4 times and different experimental approaches were utilized in order to corroborate the data more accurately. For instance, the activation of NFkB in all the cell lines was realized by using EMSA analysis, translocation of p65 subunit of NFkB to the nucleus by immunofluorescence, I-kappa B degradation by western-blot and p50 and p65 determination in the nuclear protein extracts stimulated with either the N-terminal or C-terminal domains of PTHrP. In addition to that, these changes in NF-kB activation were related with an increased in IL-6 mRNA expression. Again, the detection of IL-6 mRNA was determined using RT-PCR and Northern-Blot analysis to corroborate the data. Something similar occurred with the other experiments presented in this paper. Finally, i would like to add that this paper has been referenced many times and the results obtained in this manuscript have been corroborated by many other labs. Best regards Carlos , 
 #6 I appreciate the difficulty of responding to these concerns without access to the original data. *Hypothetically*, if you were to encounter a published image like the one illustrated below (from #3), in a 20-year old paper by other authors, what do you think should happen? In this case, it is crystal clear that the first and last lanes are derived from the same source, as all of the background noise is an exact match. ![file] To be clear, this concern is not about whether the conclusions of the paper have been replicated or cited by others. This concern is solely about the authenticity of this particular image. How do you think concerns of this type should be addressed? , 
 Thanks for the comment Actinopolyspora biskrensis. If i had the original images, i could find the correct scanned blot from the correct experiment. Furthermore, i could determine the localization (from the same blot, of course), of the last lane of the image indicated. In these experiments of IkB degradation, the band diminished during the first 10-15 minutes and afterwards the level increased (figures 3, 10 and 12 from the paper). The band intensity of the last lane was always very similar to the first one (corresponding to the basal condition). Then, probably it was used (by mistake and not intentionally) another and very similar band from the same blot. I do not know if i explained correctly what i wanted to say. Best regards Carlos , 
 #8 Thank you again. My question is more general: what do you think should happen in these *types* of cases (as this is not unique)? Choices: Nothing? Contact the journal and let editors decide? Independent investigation? An expression of concern (or other editorial note)? Retraction? Something else? , 
 Thanks again for your comment Actinopolyspora biskrensis. I do not know what is the best option. My idea of scientific research is focused on the advance of knowledge for understanding the cause of the diseases and trying to generate new treatments or better ones for curing people. I think that one of the facts that generates advance in science is the reproducibility of the results, obtained in different laboratories independently. If our results have been corroborated by others (as it is the case), and is not possible to obtain the original data (as it is the case as well), and many years have passed, scientific community will  decide . I mean that, if nobody obtain similar results of your publication, very few people will cite your work and, as time passes, your work will  disappear for people in science. So, i think that science can be auto-regulated by the scientific community. This is my modest and honest opinion. Thanks again Actinopolyspora biskrensis Best regards Carlos ,