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Down-regulation of neuropathy target esterase by protein kinase C activation with PMA stimulation

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Abstract

Neuropathy target esterase (NTE) was originally identified as the primary target site of those organophosphorus compounds that induce delayed neuropathy in human and some animals. Here we examined the role of protein kinase C (PKC) in the regulation of the NTE activity in mammalian cells. Six-hour exposure of human neuroblastoma SK-N-SH cell to a PKC activator phorbol 12-myristate 13-acetate (PMA) decreased the activity of NTE, and this effect was blocked by the PKC inhibitor staurosporine. These results suggest that PKC down-regulates the activity of NTE. NTE protein levels were down-regulated by PMA-stimulation as detected by Western blot analysis using the NTE-specific antibody, which resulted from down-regulation of NTE mRNA level as verified by real-time reverse transcription polymerase chain reaction (RT-PCR). However, there were no changes in the activity or protein levels of stable expression of NTE esterase activity domain (NEST) in SK-N-SH cells and transient expression of full-length NTE construct in COS7 cells driven by cytomegalovirus (CMV) promoter rather than by the cell’s own one, despite the absence or presence of PMA stimulation. Together, these findings suggest that stimulation with PMA reduces the expression of NTE mRNA levels but does not affect the exogenous promoter-driven NTE expression in mammalian cells.

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Abbreviations

cAMP:

Cyclic AMP

DMEM:

Dulbecco’s modified Eagle’s medium

EDTA:

Ethylenediaminetetraacetic acid

ECL:

Enhanced chemiluminescence

ER:

Endoplasmic reticulum

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GPC:

Glycerophosphocholine

NEST:

NTE esterase activity domain

NP40:

Nonidet P-40

NTE:

Neuropathy target esterase

OPIDN:

OP-induced delayed neuropathy

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PKC:

Protein kinase C

PMA:

Phorbol 12-myristate 13-acetate

PtdCho:

Phosphatidylcholine

PV:

Phenyl valerate

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30470228) and the National High Technology Research and Development Program of China (863 Program) (2006AA06Z423). The authors would like to thank Dr. Paul Glynn for providing the NTE cDNA clone D16, Dr. Yan-Lin Wang for providing the COS7 cell, Prof. Xin-Fu Leng for technical guidance in the synthesis of mipafox and phenyl valerate, and Dr. Susan Nozell for polishing the English.

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Authors and Affiliations

  1. Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuanxilu Road, Beijing, 100080, P.R. China

    Rui Chen, Ping-An Chang, Ding-Xin Long, Lin Yang & Yi-Jun Wu

  2. Graduate School of the Chinese Academy of Sciences, Beijing, 100039, P.R. China

    Rui Chen, Ding-Xin Long & Lin Yang

  3. College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing, 400065, P.R. China

    Ping-An Chang

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  1. Rui Chen
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  2. Ping-An Chang
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Corresponding author

Correspondence to Yi-Jun Wu.

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Rui Chen and Ping-An Chang contributed equally to the article.

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Chen, R., Chang, PA., Long, DX. et al. Down-regulation of neuropathy target esterase by protein kinase C activation with PMA stimulation. Mol Cell Biochem 302, 179–185 (2007). https://doi.org/10.1007/s11010-007-9439-0

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  • DOI: https://doi.org/10.1007/s11010-007-9439-0

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