Regulation of ERK1/2 phosphorylation by acute and chronic morphine - implications for the role of cAMP-responsive element binding factor (CREB)-dependent and Ets-like protein-1 (Elk-1)-dependent transcription; small interfering RNA-based strategy.
Extracellular signal-regulated kinases (ERKs) have been shown to be activated by opioids and functionally linked to addiction. Morphine-associated changes in ERK activity seem to be the characteristic features of opioid action. In this study, we observed a rapid and severe increase in ERK1/2 activity after a 5 min morphine treatment of HEK-MOR cells (transfected with the rat mu-opioid receptor MOR1) expressing mu-opioid receptor. Cellular adaptations to chronic (72 h) morphine treatment were manifested by a slight and sustained increase in ERK1/2 activity. Withdrawal caused by an opioid receptor antagonist - naloxone - attenuated phosphorylation of ERK1/2. Little information is available on the precise mechanism of ERK activity regulation. Using RNA interference technology, we generated stably transfected cells with silenced expression of cAMP-responsive element binding factor (CREB) and Ets-like protein-1 (Elk-1) transcription factors, which are known targets for activated ERK1/2. In these cells, ERK1/2 activity regulation was altered. Silencing of CREB or Elk-1 significantly increased ERK activation observed after 5 min of morphine stimulation. The initial level of activated ERKs in these cells was also augmented. Moreover, the cellular response to withdrawal signals and chronic opioid treatment was diminished. These differences suggest that both CREB-dependent and Elk-1-dependent transcription contribute to the expression of proteins regulating morphine-induced ERK activity (particular phosphatases, upstream kinases or their activatory proteins).
Ligeza
Agnieszka
A
Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland.
Wawrzczak-Bargiela
Agnieszka
A
Kaminska
Dorota
D
Korostynski
Michal
M
Przewlocki
Ryszard
R
D015153:Blotting, Western; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009020:Morphine / Q000494:pharmacology*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000502:physiology*; D050807:ets-Domain Protein Elk-1 / Q000502:physiology*
D017362:Cyclic AMP Response Element-Binding Protein; D034741:RNA, Small Interfering; D050807:ets-Domain Protein Elk-1; D009020:Morphine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3