Enhanced expression of WD repeat-containing protein 35 via CaMKK/AMPK activation in bupivacaine-treated Neuro2a cells
Authors: Guo-Gang Feng
Lei Huang
Masahiko Gosho
Misako Harato
Zhong-yuan Xia
Fumio Kondo
Yoshihiro Fujiwara
Shoshiro Okada
Naohisa Ishikawa
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: bupivacaine; AMPK; p38 MAPK; WDR35; CaMKK
Affiliations: Department of Pharmacology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan; Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China., Department of Pharmacology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan., Advanced Medical Research Center, Aichi Medical University, Nagakute, Aichi, Japan., Department of Anesthesiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan., Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China.

Abstract:  We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression. 

Retracted: No

PubPeer comments:
 2019 Expression of concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0213041 Expression of Concern: Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK AMPK Activation in Bupivacaine-Treated Neuro2a Cells The PLOS ONE Editors Published: February 22, 2019https: doi.org 10.1371 journal.pone.0213041 Following the publication of [1] concerns were raised in the following figure panels: Fig 1B AMPKu03b1 panel appears similar to Fig 2A AMPKu03b1 panel Fig 1B P-AMPKu03b1 panel contains vertical discontinuities across lanes Fig 3A P-AMPKu03b1 panel contains vertical discontinuities across lanes Fig 3A P-p38 panel contains a vertical discontinuity after lane 9 Fig 5A P-AMPKu03b1 panel contains a vertical discontinuity after lane 2 Fig 6B WDR35 panel contains a vertical discontinuity after lane 2 The authors explained that an incorrect image was used during preparation of Fig 1 and have provided a replacement image for the Fig 1B AMPKu03b1 panel. In the case of the Fig 1B P-AMPKu03b1 panel, the authors explained that samples were run in duplicate for Bupivacaine treatment and they removed the duplicate samplesu2019 data in preparing the figure. However, the raw data provided by the authors suggests that the results across these duplicate samples were variable and those shown in the figure were not representative of the overall results. The authors also noted that they spliced out lanes from the original image in preparing Fig 2A, and rearranged data in Fig 5A P-AMPKu03b1 panel for the purpose of presentation. For both of these panels, the authors provided the original blot data supporting the blots of concern. Individual-level data underlying all the graphs in the article are available. However, authors have been unable to provide raw unadjusted blots underlying panels of concern in Figs 3A and 6B and are therefore not compliant with our data availability policy. In light of the image concerns and the unavailability of data underlying the findings, the PLOS ONE Editors post this Expression of Concern. Reference 1. Huang L, Kondo F, Gosho M, Feng G-G, Harato M, Xia Z-y, et al. (2014) Enhanced Expression of WD Repeat-Containing Protein 35 via CaMKK AMPK Activation in Bupivacaine-Treated Neuro2a Cells. PLoS ONE 9(5): e98185. https: doi.org 10.1371 journal.pone.0098185 pmid:24859235 , 
 Concerns about Figure 1, as reported to the journal in May 2014. ![file] ,