enkephalin--ala(2)-mephe(4)-gly(5)- and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Opioid receptor agonists induce broad immunomodulatory activity, which substantially alters host defense and the inflammatory response. Previous studies have shown that the MOR selective agonist DAMGO has the capacity to increase the expression of the proinflammatory chemokines CCL2, CCL5, and CXCL10 in human PBMCs. NF-κB is a transcription factor that plays a pivotal role in innate and adaptive immune responses. We report that NF-κB is a vital player in the DAMGO-induced, MOR-mediated regulation of chemokine expression. Results show that NF-κB inhibitors prevent the induction of CCL2 expression in response to DAMGO administration and that the NF-κB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to MOR activation. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation, and this kinase is essential for NF-κB activation as well as CCL2 expression and transcriptional activity. Finally, ChIP analysis shows that DAMGO administration induces binding of p65 to the enhancer region of the CCL2 promoter. These data are consistent with the notion that MOR activation promotes a proinflammatory response, which involves NF-κB activation. Our results also suggest a significant and novel role for PKCζ as an essential participant in the MOR-mediated regulation of proinflammatory chemokine expression.

Topics: Aldehydes; Analgesics, Opioid; Cells, Cultured; Chemokine CCL2; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; HEK293 Cells; Humans; Leupeptins; NF-kappa B; Nitriles; Protein Kinase C; Protein Kinase Inhibitors; Receptors, Opioid, mu; Sulfones

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ∼60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα(i/o) and Gα(q) proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα(i/o)-coupled DOR and the Gα(q)-coupled M(3) muscarinic receptor (M(3)R) was increased but not signaling of the α(2) adrenergic receptor or bradykinin BK(2) receptor, suggesting the development of cross-talk between the DOR and M(3)R involving RGS4.

Topics: Acetylcysteine; Analgesics, Opioid; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; HEK293 Cells; Humans; Leupeptins; Naloxone; Narcotic Antagonists; Pertussis Toxin; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptor, Muscarinic M3; Receptors, Adrenergic, alpha-2; Receptors, Bradykinin; Receptors, Opioid, delta; Receptors, Opioid, mu; RGS Proteins; Signal Transduction; Time Factors; Ubiquitination