Dynamic association of p300 with the promoter of the G protein-coupled rat delta opioid receptor gene during NGF-induced neuronal differentiation.

The G protein-coupled delta opioid receptor (DOR) plays a critical role in pain control. Emerging evidence shows that DOR also plays a role in neuronal differentiation and survival. Nerve growth factor (NGF) is known to be critical for the development and maintenance of the central and peripheral nervous systems. Our previous studies have shown that sustained activation of NGF/PI3K/Akt/NF-kappaB signaling is essential for NGF-induced dor gene expression during neuronal differentiation and that the epigenetic modifications at histone 3 lysine 9 temporally correlate with the dor gene transcription. In this study, we cloned the rat dor gene promoter and identified an NGF-responsive region similar to that from the mouse dor gene promoter. We further identified p300, a known NF-kappaB binding partner with intrinsic histone acetyltransferase activity, to be dynamically associated with the dor gene. We also found that assembling of RNA polymerase II (Pol II) at the promoter took place before NGF stimulation, indicating that p300 could only interact with preassembled Pol II at the promoter after NGF stimulation. Taken together, these results implicate that preassembly of the Pol II preinitiation complex, sustained activation of PI3K/Akt/NF-kappaB signaling, and dynamic p300 association at the promoters sequentially is one of the mechanisms of induction of the late phase genes during NGF-induced neuronal differentiation.

Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth.

Morphine is used to treat pain in several medical conditions including cancer. Here we show that morphine, in a concentration typical of that observed in patients' blood, stimulates human microvascular endothelial cell proliferation and angiogenesis in vitro and in vivo. It does so by activating mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation via Gi/Go-coupled G protein receptors and nitric oxide in these microvascular endothelial cells. Other contributing effects of morphine include activation of the survival signal PKB/Akt, inhibition of apoptosis, and promotion of cell cycle progression by increasing cyclin D1. Consistent with these effects, morphine in clinically relevant doses promotes tumor neovascularization in a human breast tumor xenograft model in mice leading to increased tumor progression. These results indicate that clinical use of morphine could potentially be harmful in patients with angiogenesis-dependent cancers.

Inhibition of PI3K/Akt signaling: an emerging paradigm for targeted cancer therapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway plays a critical role in cell growth and survival. Dysregulation of this pathway has been found in a variety of cancer cells. Recently, constitutively active PI3K/Akt signaling has been firmly established as a major determinant for cell growth and survival in an array of cancers. Blocking the constitutively active PI3K/AKT signaling pathway provides a new strategy for targeted cancer therapy. Thus, inhibitors of this signaling pathway would be potential anticancer agents, particularly for cancer cells whose survival and growth are dominated by constitutively active PI3K/Akt signaling. This review describes the current understanding of small molecule drugs targeting this pathway both in vitro and in vivo. Inhibitors and functions of the upstream and downstream molecular targets of the PI3K/Akt pathway are discussed in the context of using the inhibitors to block this pathway for targeted cancer therapy. Special emphasis is placed on the following targets: receptor tyrosine kinases, PI3K, Akt, and the mammalian target of rapamycin. While the molecular therapeutic strategy holds great promise for the treatment of a variety of cancers, few small molecule inhibitors with potential high therapeutic indexes are available. Thus, new inhibitors with high selectivity, bioavailability, and potency are greatly needed. Novel approaches toward the development of PI3K/Akt pathway inhibitors as anticancer therapeutics are discussed in detail, with emphasis on chemical genetics-based and structure-based drug design.

Naphthalene dicarboxaldehyde as an electrophilic fluorogenic moiety for affinity labeling: application to opioid receptor affinity labels with greatly improved fluorogenic properties.

To develop ligands with fluorogenic properties amenable for following the kinetics of cross-linking to receptors, a naphthalene dicarboxaldehyde moiety has been attached to an opiate pharmacophore 2 and evaluated in mu-opioid receptors. The fluorescence of the benzo[f]isoindole formed upon cross-linking of mu-opioid receptors by 2 permitted the time-course of covalent bonding to be followed. This demonstrated proof-of-concept suggests the usefulness of naphthalene dicarboxaldehyde-containing affinity labels as kinetic probes.

Mu opioid receptor from the zebrafish exhibits functional characteristics as those of mammalian mu opioid receptor.

The functional characterization of the mu opioid receptor from the zebrafish (zfMOR) is reported here. After transfection in HEK-293 cell line, using both peptidergic and nonpeptidergic opioid ligands in the competition and saturation-binding experiments, in addition to the functional assays of (35)S-GTPgammaS-binding assays and intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) level determinations, we demonstrate that zfMOR exhibits a pharmacological profile similar to that of the mammalian MOR. Besides, the internalization process of zfMOR after opiate agonist treatment (morphine, DAMGO, etorphine) resembles the pattern observed for its mammalian counterpart. These similarities suggest that the zebrafish is a good model for the study of the opioid effects in development.

The first and third intracellular loops together with the carboxy terminal tail of the delta-opioid receptor contribute toward functional interaction with Galpha16.

Opioid peptides exert their regulatory effects on both central and peripheral nervous systems via multiple opioid receptors that are linked to seemingly identical sets of guanine nucleotide-binding regulatory proteins (G proteins). In contrast to the mu-opioid receptor, the delta-opioid receptor can efficiently stimulate phospholipase C via G16. We used a series of mu/delta-opioid receptor chimeras to examine the involvement of intracellular receptor domains in the recognition of G16. After ascertaining that the chimeras can bind opioid ligands with high affinity and elicit inhibition of adenylyl cyclase, COS-7 cells were cotransfected with cDNAs encoding Galpha16 and a mu/delta-opioid receptor chimera and assayed for [D-Ala2,D-Leu5]enkephalin-induced stimulation of phospholipase C. Our results indicate that (i) the carboxy terminal tail of the delta-opioid receptor is necessary but insufficient for conferring coupling to Galpha16, (ii) the third inner loop together with the carboxy terminal tail of the delta-opioid receptor can provide sufficient contact domains for Galpha16, and (iii) the first inner loop of the delta-opioid receptor, in particular Leu80, as well as the fifth transmembrane domain and/or the third extracellular loop may also contribute in defining the fidelity of interaction between the delta-opioid receptor and Galpha16. These results indicate that efficient coupling of the delta-opioid receptor to Galpha16 requires the participation of most of the intracellular regions, including the first intracellular loop.