ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation
Authors: I-Ta Lee
Shin-Ei Cheng
Wan-Ling Wu
Chih-Chung Lin
Li-Der Hsiao
Chuen-Mao Yang
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: COX-2; PGE(2); ATP; P2 purinoceptors; lung inflammation
Affiliations: Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.

Abstract:  Background: Up-regulation of cyclooxygenase (COX)-2 and its metabolite prostaglandin E(2) (PGE(2)) are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2) release remain unclear. Principal findings: Here, we showed that ATPŒ≥S induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin), PKC (G√∂6983, G√∂6976, Ro318220, and Rottlerin), ROS (Edaravone), NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin], Jak2 (AG490), and STAT3 [cucurbitacin E (CBE)] and transfection with siRNAs of PKCŒ±, PKCŒπ, PKCŒº, p47(phox), Jak2, STAT3, and cPLA(2) markedly reduced ATPŒ≥S-induced COX-2 expression and PGE(2) production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPŒ≥S. Moreover, ATPŒ≥S-induced ROS generation and p47(phox) translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPŒ≥S stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, G√∂6983, G√∂6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. Significance: Taken together, these results showed that ATPŒ≥S induced COX-2 expression and PGE(2) production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2) signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies. 

Retracted: Yes

PubPeer comments:
 Figure 4F. This issue has been reported to the journal in May 2014, but has not yet been addressed. * Red arrows: The Membrane PKCalpha and Membrane PKCiota panels appear to share three lanes, if one of the two panels is shown in mirror image.  , 
 Figure 4A and 5A. This issue has been reported to the journal in May 2014, but has not yet been addressed. * Blue boxes: The top COX-2 panel in Figure 4A (PPADS added) looks similar to the bottom COX-2 panel in Figure 5A (CBE added).  , 
 Figure 5F. This issue has been reported to the journal in May 2014, but has not yet been addressed. * Orange boxes: The Jak2 panel in the top half of the figure looks similar to the STAT3 panel in the bottom half of the figure. * Magenta boxes: The actin panel in the top half of the figure looks similar to the actin panel in the bottom half of the figure.  , 
 Figure 2E. This issue has been reported to the journal in May 2014, but has not yet been addressed. * Green boxes: The 0min and 5min panels appear to show an overlap. * Light blue boxes: The 30min and 60min panels appear to show an overlap.  , 
 #1 Dear Dr. Bik, thank you very much for pointing out this mistake. We have checked our original figures and found that we uploaded wrong figure by mistake. This did not affect our results and conclusion. We will contact the editor of the journal to resubmit the correct figures soon. , 
 Retraction, 19 May 2022: https: journals.plos.org plosone article?id=10.1371 journal.pone.0268877  Following the publication of this article [1], concerns were raised regarding results presented in multiple figures. Specifically, * In Fig 2E, the following panels appear to partially overlap despite representing different experimental conditions: u25cb The 0 min and 5 min panels. u25cb The 30 min and 60 min panels. * In Figs 2F, 3B and 4D, the Basal and ATPu03b3S control panels appear similar. * The following western blot data appear similar despite representing different conditions: u25cb Lanes 3u20135 of the p47phox panel in Fig 2G, lanes 1u20133 of the PKCu03b1 membrane panel in Fig 4F flipped horizontally, and lanes 1u20133 of the PKCu03af membrane panel in Fig 4F. u25cb Lane 2 of the p47phox panel in Fig 2G and lane 4 of the PKCu03b1 membrane panel in Fig 4F, flipped horizontally. u25cb Lane 6 of the p47phox panel in Fig 2G and lane 4 of the PKCu03af membrane panel in Fig 4F. u25cb Lane 1 of the GAPDH panel and lane 4 of the Gu03b1s panel in Fig 4F. u25cb The COX-2 panel for PPADS in Fig 4A and the COX-2 panel for CBE in Fig 5A. u25cb In Fig 5F, the Jak2 and STAT3 panels, and the u03b2-actin for Jak2 and u03b2-actin for STAT3 panels. u25cb In Fig 6A (right side), lanes 1u20132 of the T-STAT3 (CE) panel and lanes 6u20137 of the T-STAT3 (NE) panel. The corresponding author stated that the 5 min and 60 min panels in Fig 2E were incorrect. They provided an updated figure with these panels replaced and raw image data underlying this experiment. The corresponding author stated that multiple western blot experiments were performed and imaged at the same time. They acknowledged that errors occurred in the preparation of the figures and annotation of raw images, and apologized for these errors. The corresponding author accepted that the issues with the published figures cannot be fully resolved, and requested that the article be retracted. In light of the concerns affecting multiple figure panels and the errors in the raw underlying data that question the reliability of these data, the PLOS ONE Editors retract this article. ITL, CCL, WLW, LDH and CMY agreed with the retraction and apologize for the issues with the published article. SEC either did not respond directly or could not be reached. ,