The Orai-1 and STIM-1 complex controls human dendritic cell maturation
Authors: Yvon Lebranchu
David Crottès
Jérémy Fauconnier
Anthony Delalande
Romain Félix
Jean-Yves Le Guennec
Florence Velge-Roussel
Journal: PloS one (2013)
Discipline: Multidisciplinary
Keywords: Ca(2+) signaling; dendritic cells; CRAC channels; maturation; cytokine secretion
Affiliations: EA 4245 Cellules Dendritiques, Immunomodulation et Greffes, Université François Rabelais, IFR-136 Agents Transmissibles et Infectiologie, UFR de Médecine, Tours, France.

Abstract:  Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins. 

Retracted: No

PubPeer comments:
 Figure 6C * Yellow boxes: The top and bottom panel appears to show an overlap. ![file] , 
 April 2020 Expression of Concern, which reads: *After this article [1] was published, concerns were raised about Fig 6C:* *The upper portion of the top blot appears similar to the lower blot. These panels are used to demonstrate the efficiency of siRNA knockdown, but the difference between the control and experimental data in these blots is modest and the blot experiment did not include internal controls to demonstrate the relative protein abundance across lanes. The authors provided image data in support of these results. They commented that the same number of cells is represented in each lane but they did not conduct control blots (e.g. actin) to assess relative protein levels across lanes in each blot. The image data provided (S1 File) do not match the results included in the published figure and do not demonstrate clear differences in protein expression in the experimental versus control siRNA samples.* *Regarding the relative protein abundance in siRNA knockdown versus control samples, the authors acknowledged that the western blot quality was poor and they referred to supporting results in Fig 6A and 6B which indicate functional differences between experimental and control siRNA samples.* *The concerns about western blot data reporting in Fig 6C are not fully resolved, and questions remain about the evidence supporting siRNA-mediated knockdown of protein expression. The articleu2019s conclusions about roles of STIM1 and Orai1 rely on the siRNA knockdown model for which Fig 6C provides key validation data. As such, the unresolved issues have implications for whether the results reported in this article adequately support these conclusions. For these reasons, the PLOS ONE Editors issue this Expression of Concern.* *FVR noted that the authors stand by the results in the article.* *All raw data are available except those for Fig 5C and for the flow cytometry (.fcs files for Figs 1, 3 and 7) and biophysics experiments (Figs 2 and 6A).* *The authors provided the following product information for reagents used in this study: siRNA ORAI-1 (h), Santa Cruz Biotechnology #sc-76001; siRNA STIM-1 Santa Cruz Biotechnology #sc-76589; anti-ORAI1 antibody, Santa Cruz Biotechnology #A47359.* https: journals.plos.org plosone article?id=10.1371 journal.pone.0231469 , 
 2020 Expression of Cocern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0231469 After this article [1] was published, concerns were raised about Fig 6C: The upper portion of the top blot appears similar to the lower blot. These panels are used to demonstrate the efficiency of siRNA knockdown, but the difference between the control and experimental data in these blots is modest and the blot experiment did not include internal controls to demonstrate the relative protein abundance across lanes. The authors provided image data in support of these results. They commented that the same number of cells is represented in each lane but they did not conduct control blots (e.g. actin) to assess relative protein levels across lanes in each blot. The image data provided (S1 File) do not match the results included in the published figure and do not demonstrate clear differences in protein expression in the experimental versus control siRNA samples. Regarding the relative protein abundance in siRNA knockdown versus control samples, the authors acknowledged that the western blot quality was poor and they referred to supporting results in Fig 6A and 6B which indicate functional differences between experimental and control siRNA samples. The concerns about western blot data reporting in Fig 6C are not fully resolved, and questions remain about the evidence supporting siRNA-mediated knockdown of protein expression. The articleu2019s conclusions about roles of STIM1 and Orai1 rely on the siRNA knockdown model for which Fig 6C provides key validation data. As such, the unresolved issues have implications for whether the results reported in this article adequately support these conclusions. For these reasons, the PLOS ONE Editors issue this Expression of Concern. FVR noted that the authors stand by the results in the article. All raw data are available except those for Fig 5C and for the flow cytometry (.fcs files for Figs 1, 3 and 7) and biophysics experiments (Figs 2 and 6A). The authors provided the following product information for reagents used in this study: siRNA ORAI-1 (h), Santa Cruz Biotechnology #sc-76001; siRNA STIM-1 Santa Cruz Biotechnology #sc-76589; anti-ORAI1 antibody, Santa Cruz Biotechnology #A47359. ,