Neuronal differentiation dictates estrogen-dependent survival and ERK1/2 kinetic by means of caveolin-1
Authors: Floriana Volpicelli
Massimiliano Caiazzo
Umberto di Porzio
Luca Colucci-D&#x27;Amato
Bruno Moncharmont
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: Estrogens; CNS; development; mature functions; proliferation
Affiliations: Department of Pharmacy, University of Naples "Federico II", Naples, Italy; Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR, Naples, Italy., Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR, Naples, Italy; Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland., Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy., Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR, Naples, Italy., Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy; C.I.R.N., Inter-University Center for Research in Neuroscience, Naples, Italy.

Abstract:  Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival. 

Retracted: Yes

PubPeer comments:
 ![file] , 
 ![file] , 
 #1 Also note the duplication between first and last lanes of P-ERK1 2 panel of Fig 4C: ![file] , 
 #2 Also note apparent duplication of ERK1 2 data between Figs 5A and 5C (in flipped orientation), plus a duplicated band within Fig 5C: ![file] , 
 2019 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0224493 After publication of this article [1], concerns were raised about results reported in Figures 1, 4, and 5: In Figure 1A, there appears to be an image splicing line between lanes 2 and 3 of the u03b2-actin blot. In Figure 4C, the P-ERK1 2 and ERK1 2 panels do not include the same numbers of lanes, and similarities were noted between data shown in lanes 1 and lane 7 of the P-ERK1 2 blot. There also appear to be vertical discontinuities in Figure 4C, P-ERK1 2 panel after lanes 2 and 6. In the P-ERK1 2 panel of Figure 5A, similarities were noted between the data shown in lanes 1 and 2, and there appears to be a vertical discontinuity between lanes 2 and 3. In the ERK1 2 panel of Figure 5C, similarities were noted between the data shown in lanes 1 and 4, and there appears to be a vertical discontinuity after lane 5. Similarities were noted between data shown in Figure 5A, ERK1 2 panel lanes 1u20135, and Figure 5C, ERK1 2 panel lanes 6u20132. In Figure 5C, P-ERK1 2 panel, there appear to be vertical discontinuities after lanes 3, 4, and 5, and the background appears notably different in lanes 5 and 6 compared to lanes 1u20133. For Figure 1, the authors noted that the A1 and mesPC data were obtained using different gels blots, and that the corresponding experimental and u03b2-actin data were obtained from the same blot in each case. The authors provided raw image data to support the Figure 4 results. These data clarified that lanes were rearranged in preparing the ERK1 2 panel in Figure 4A and the P-ERK1 2 panels in Figure 4A and 4C, and that lane 7 in each of these panels should have been labelled as exposed to both estradiol (E2) and ICI 182u2013780. For the ERK1 2 panel of Figure 4C, the E2 + ICI 182u2013780 data are in the third lane, an additional timepoint (20 min) was included in error as the fourth lane, and lanes 5u20138 are the data corresponding to the lane 3u20136 labels. The raw data indicated that the wrong control data (control without ethanol) were included in lane 1 of the P-ERK1 2 panels of Figure 4A and 4C. The authors commented that similarities between lanes 1 and 7 of the P-ERK1 2 panel in Figure 4C may reflect similarities in expression levels in the two samples, or could be due to an error in the figure preparation. Per our editorial assessment the pixel data in these lanes are more similar than would be expected for different data, and the E2 + ICI 182u2013780 data on the raw blot image did not appear to match the data in the published figure. Hence, concerns about the lane 7 P-ERK1 2 data in Figure 4C remain unresolved. For Figure 5, the authors provided quantification data for the panels in question and claimed that the data raised as similar were not the same, although per our editorial assessment the pixel data in the affected lanes are more similar than would be expected for different data. The original blot images are unavailable and so we were unable to resolve the concerns. The underlying data were not provided with this article, although the Data Availability statement reads, u201cAll relevant data are within the paper and its Supporting Information files.u201d The authors commented that the underlying data are not available for Figures 1, 2A, 5, S1, S2, S3, and S6. For Figure 3, quantitative data are available but the underlying blot images are not. Underlying data supporting other results reported in the article are available from the authors, as are FACS data to support the u201cdata not shownu201d statement in the Results section. In light of the unresolved concerns about Figures 4 and 5 as well as the unavailability of some data needed to support this articleu2019s results, the PLOS ONE Editors retract this article. It was noted after publication that the Academic Editor who handled this articleu2019s peer review was affiliated with the same institution as one of the authors. We regret that this was not identified and addressed prior to the articleu2019s publication. All authors disagreed with the retraction. Reference 1.Volpicelli F, Caiazzo M, Moncharmont B, di Porzio U, Colucci-Du2019Amato L (2014) Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1 2 Kinetic by Means of Caveolin-1. PLoS ONE 9(10): e109671. https: doi.org 10.1371 journal.pone.0109671 pmid:25350132 , 
 https: retractionwatch.com 2019 10 31 plos-one-realizes-an-academic-editor-had-a-conflict-of-interest-that-the-publisher-says-it-now-tries-harder-to-avoid , 
 Not only do we strongly disagree with the decision to retract the paper, but we would like the readers to learn, at least, some of the main points of the reviewing process that has led PlosOne to retract the paper. Thus, we attached a link [ ] containing a more detailed description of the events occurred, as well as, the data provided by us. Briefly, there are two figures that are disputed: fig 4 and 5 and then PlosOne rose the question that the u201cAcademic Editor who handled this articleu2019s peer review was affiliated with the same institution as one of the authorsu201d. Concerning Fig. 4, we were able to provide the original data showing that a timepoint was omitted whereas the controls were left as such (see link attached). Thus, it was just a mere mistake following the rearrangement of the lanes. Concerning Fig 5, which unfortunately we were not able to provide, the journal says:u201cper our editorial assessment the pixel data in the affected lanes are more similar than would be expectedu201d. Indeed, we had provided evidence that the bands are not the same by applying an ImageQuant software analysis of the pixels (see link attached). It is inconsistent with common sense that a duplicated data is similar, since a duplicated data should be identical. Thus, the decision is taken in the absence of evidence. Not a commendable action for a science journal claiming to comply with evidence! Concerning the Academic Editor, the Journal noted u201cafter publication that the Academic Editor who handled this articleu2019s peer review was affiliated with the same institution as one of the authors. We regret that this was not identified and addressed prior to the articleu2019s publicationu201d. We are extremely upset for such an astonishing statement after 6 yrs since the paper has been published. Normally, this is part of the job of the staff to choose the editors referees. The choice of the Academic editor was made by the Journal, not even suggested by us!! According to the present comments of the people who handled our paper, we should have rejected their own choice, who, indeed, carried out a very accurate review so that we had to add 7(!) new figures to the 7 already presented. Thus, since a very small portion of the paper is disputed (1 figure out of 14 and 1 lane out of 7) and since we are absolutely confident about our data, we proposed drs Heber and Renu00e8e Hoch, who handled the paper, to perform ex novo experiments containing the data of figs 4 and 5 and to publish a correction, also considering that the kinetics included in fig 4C was also present in other experiments (fig 6C), giving solidity to our data. This would have been perfectly consistent with the guidelines contained in COPE (see attached link) that the Journal complies with. On the contrary, the editor-in-chief invokes that since 2019 they require u201cto submit original uncropped adjusted images supporting all blot and gel results reported in an articleu2019s figures and supporting information filesu201d, although the article was published in 2014. The retroactive way to apply procedures is not a straightforward way to act. Moreover, they have also ignored the COPE guidelines! In addition, concerning the reviewing process, some statements and requests appear astonishing i.e. u201cPlosOne does not allowed reliance on reference on unshown datau2026.thus provide the original underlying data supporting the sentenceu2026u201d. In spite of the fact that we were able to find and provide the data (a FACS analysis of estrogen-starved cells), again, we think this is a strange, not transparent procedure after 5 yrs since the paper has been published. In conclusion, on the whole, we think that the reviewing process has been superficial, lacking in transparency, careless and has come to a decision that we, believe is unfair, out of the context, and not honest, (see link attached) rejecting the chance of a correction as asked by us being confident 100% about our data. Thus, although the research project whose data are included in the rejected manuscript is over, we have planned to re-submit a new version of the paper to another journal. As a consequence of this event, we will be careful not to submit anymore our data to PlosOne. Luca Colucci-Du2019Amato, Floriana Volpicelli, Massimiliano Caiazzo, Bruno Moncharmont, Umberto di Porzio , 
 #7 The paper has been retracted by the journal. , 
 *Concerning Fig 5, which unfortunately we were not able to provide, the journal says:u201cper our editorial assessment the pixel data in the affected lanes are more similar than would be expectedu201d. Indeed, we had provided evidence that the bands are not the same by applying an ImageQuant software analysis of the pixels (see link attached). It is inconsistent with common sense that a duplicated data is similar, since a duplicated data should be identical. Thus, the decision is taken in the absence of evidence. Not a commendable action for a science journal claiming to comply with evidence!* The bands are more indeed more similar than might be expected, and appear to be two *exposures* of the same bands with partial flip. The set of bands in A has a clearly darker background. Your ImageQuant analysis is not suitable to disprove the former. Comparing largely saturated signal which has  topped out with unsaturated bands and finding *surprise* they are different will not ever prove uniqueness. It is your duty to provide data to back up your papers contentions, if they are in doubt. You have reversed normal expectations of responsibility to blame the journal for retracting based on your failure to provide it. , 
 #7 As the Bard said, the authors * doth protest too much, methinks *. These protestations are not convincing. The irregularities noted above in this thread are clearly evident to any objective observer. * From post #2: in Fig 5A, top panel, lanes 1 and 2 appear to be duplicates. * From post #3: in Fig 4C, top panel, lanes 1 and 7 appear to be duplicates. * From post #4: there appears to be duplication of ERK1 2 data (horizontally flipped) in Figs 5A and 5C, plus a duplicated band within Fig 5C. These are all quite clear from simple visual inspection. The authors argue that  ImageQuant analysis of Fig 5 indicates the suspected duplicates are not identical, and they state that * It is inconsistent with common sense that a duplicated data is similar, since a duplicated data should be identical. * This argument is neither logical nor credible. If one duplicates an image and makes any minor change (e.g., lighten darken or flip rotate the image), by necessity the images are no longer identical. But whether they are precisely identical is not the appropriate criterion to apply -- instead, the key issue is whether the images are likely to have a different origin or if the similarities are so extensive that they are likely to have a shared origin (see the P.S. below for further information and citation). In the examples above, the answer is clear. Thus, the journal made the correct assessment and the right decision. In contrast, the authors statement attempts to downplay serious issues, and fails to accept responsibility. Note that there are related concerns in other papers by some of the same authors: https: pubpeer.com publications 11BAE73B53B4ED62DC0D03F00437C0 https: pubpeer.com publications 812E5C95DA10CF0831016228F930CF https: pubpeer.com publications A8F2B88889CB2BC4D2DCD9B0078EF9 * P.S., For further information about the method for judging same vs. different origins of similar images, see the following link to an affidavit by Dr. John Krueger, former official of the U.S. Office of Research Integrity (ORI), which emphasizes repeatedly that ** two images need not be pixel-for-pixel matches in order to conclude that they depict the same experiment ** and ** Instead, the question is whether there are characteristic features unexpectedly in common between the images that indicate that they are u201ctoo similar to be different.u201d ** In particular, in pages 8-10, see points numbered 15, 17, 18, and 20. https: www.aclu.org sites default files assets krueger_affidavit.pdf , 
 #7 One wonders, is there any chance that, despite the protestations from the authors, there were other irregularities that went undiscovered by previous commenters here or by the journal s investigation? Sure enough, see Figure 3. * In Fig 3A, upper panel (P-ERK1 2), remarkable similarities are evident between lanes 1 and 5. * In Fig 3C, lower panel (ERK1 2), remarkable similarities are evident between lanes 3 and 7. Each is illustrated below. The similar lanes are boxed in the original figure, and then the similar lanes have been juxtaposed and subjected to four different adjustments of gradient maps, to help visualize the similarities. **Figure 3A:** ![file] **Figure 3C:** ![file] , 
 I completely agree with the duplications observed in Figures 3, 4, and 5. There is also a duplication in Figure 6D, which has not been brought up yet, but is worth including here, as a post-mortem. ![file] , 
 #12 Good catch in Fig 6D. It emphasizes that different sets of eyes can find different things. Indeed, in retrospect I now realize that my findings in Figure 3A in post #11 were incomplete. There appear to be further duplications: * In the upper panel (P-ERK1 2), the similarities in lanes 1 and 5 ALSO extend to lanes 6 and 7. Here, there are unique spots blemishes in lanes 6 and 7, plus some unusual discontinuities between lanes 5-6 and 6-7, which could suggest post-capture alterations. Regardless, a close examination of the individual band features (especially after adjusting gradient maps) suggests that all 4 lanes have a shared origin. * In the lower panel (ERK1 2), lanes 2 and 7 appear to be duplicates. Ilustrated below. ![file] ,