Stress is a complex human experience having both positive and negative motivational properties. When chronic and uncontrollable, the adverse effects of stress on human health are considerable and yet poorly understood. Here, we report that the dysphoric properties of chronic stress are encoded by the endogenous opioid peptide dynorphin acting on specific stress-related neuronal circuits. Using different forms of stress presumed to evoke dysphoria in mice, we found that repeated forced swim and inescapable footshock both produced aversive behaviors that were blocked by a -opioid receptor (KOR) antagonist and absent in mice lacking dynorphin. Injection of corticotropin-releasing factor (CRF) or urocortin III, key mediators of the stress response, produced place aversion that was also blocked by dynorphin gene deletion or KOR antagonism. CRF-induced place aversion was blocked by the CRF 2 receptor antagonist antisauvigine-30, but not by the CRF 1 receptor antagonist antalarmin. In contrast, place aversion induced by the KOR agonist U50,488 was not blocked by antisauvigine-30. These results suggest that the aversive effects of stress were mediated by CRF 2 receptor stimulation of dynorphin release and subsequent KOR activation. Using a phospho-selective antibody directed against the activated KOR to image sites of dynorphin action in the brain, we found that stress and CRF each caused dynorphin-dependent KOR activation in the basolateral amygdala, nucleus accumbens, dorsal raphe, and hippocampus. The convergence of stress-induced aversive inputs on the dynorphin system was unexpected, implicates dynorphin as a key mediator of dysphoria, and emphasizes -receptor antagonists as promising therapeutics.

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both -opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(Ϫ/Ϫ) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4- fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) andthe KOR-dependent swim stress-induced immobility while not affecting -opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that ␤-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin--opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.

G-protein receptor kinase and ␤-arrestin mediated desensitization of the rat -opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the agonist U50,488 (100 nM) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the Gprotein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization.The use of opioid agonists to produce clinical analgesia is limited by their propensity to induce drug tolerance and dependence (1). Therefore, regulatory mechanisms responsible for opioid tolerance are of therapeutic interest. Opioid receptor desensitization and internalization are likely to play significant roles in the control of receptor signaling, but the biochemical steps underlying these mechanisms are uncertain. Agonistinduced receptor phosphorylation is thought to mediate both G-protein coupled receptor desensitization and internalization (for a recent review, see Ref.2). However, establishing a direct link between these receptor mechanisms and the phosphorylation of individual amino acid residues on the opioid receptors has been confounded by the large number of potential phosphorylation sites and the practical difficulty of identifying specific phosphorylated sites within the opioid receptors.Direct evidence of -opioid receptor (KOR) 1 phosphorylation was obtained by immunoprecipitation of 32 P-labeled KOR from guinea pig hippocampal slices treated with a KOR-selective agonist (3). This increase in KOR phosphor...

Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [ opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4 -L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAPpositive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ∼1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy.

Partial sciatic nerve ligation (pSNL) markedly increased glial fibrillary acidic protein immunoreactivity (GFAP-IR

Trigeminal nerve damage often leads to chronic pain syndromes including trigeminal neuralgia, a severely debilitating chronic orofacial pain syndrome. Options for treatment of neuropathic pain are limited in effectiveness and new approaches based on a better understanding of the underlying pathologies are required. Partial ligation has been shown to effectively mimic many of the qualities of human neuropathic pain syndromes. We have devised a mouse model of trigeminal neuralgia using a partial ligation of the infraorbital nerve (pIONL) that induces persistent pain behaviors and morphological changes in the brainstem. We found that the pIONL effectively induced mechanical allodynia lasting for over 3 weeks. Cell proliferation (BrdU), activation of astrocytes and microglia in the ipsilateral caudal medulla, and persistent satellite cell reaction in the ipsilateral ganglion were observed. Neurochemical markers CGRP, substance P were decreased in medullary dorsal horn ipsilateral to the injury side, whereas substance P receptor NK1 expression was increased after 8 days. Nerve injury marker ATF3 was markedly increased in ipsilateral trigeminal ganglion neurons at 8 days after pIONL. The data indicate that partial trigeminal injury in mice produces many persistent anatomical changes in neuropathic pain, as well as mechanical allodynia.Perspective-The study describes the development of a new mouse model of trigeminal neuropathic pain. Our goal is to devise better treatments of trigeminal pain, and this will be facilitated by characterization of the underlying cellular and molecular neuropathological mechanisms in genetically designed mice.

Transcription of the catalytic subunit of telomerase (hTERT) in keratinocytes can be induced by human papillomavirus type 16 (HPV16) E6/E6AP ubiquitin ligase through degradation of the repressor, NFX1-91. Here, we demonstrate that NFX1-91 interacts with the corepressor complex mSin3A/histone deacetylase (HDAC) at the hTERT promoter. By degrading NFX1-91, E6/E6AP changes the chromatin structure at the hTERT promoter as indicated by enhanced acetylation of histones H3 and H4 as well as dimethylation of H3K4. Knockdown of NFX1-91 by short hairpin RNA (shRNA) mimics the effect of E6 and leads to acetylation of histones H3 and H4. Conversely, knockdown of E6AP by shRNA suppresses histone acetylation at the hTERT promoter. These data demonstrate that targeted degradation of NFX1-91 by E6/E6AP dissociates the mSin3A/ HDAC complex from the hTERT promoter and induces hTERT transcription.Maintenance of telomere length by telomerase is required for sustained cell proliferation, and activation of telomerase is a critical step during cellular immortalization and malignant transformation (7). Expression of the catalytic subunit of telomerase (hTERT) is rate limiting for telomerase activity (28,30,32); thus, understanding the regulation of hTERT transcription is critical to elucidate how cells acquire immortality. The hTERT promoter contains several transcription factorbinding sites, including c-Myc (15, 42, 45), Sp1 (18,37,43), USF (12), and ER81 (13). Several studies showed that hTERT expression is minimal or absent in most somatic cells through both known transcriptional repressors, such as Mad1, menin, and SIP1 (24), and other endogenous factors demonstrated by chromosome transfer experiments, such as a putative tumor suppressor on chromosome 3p (17). Distinct repression and activation mechanisms likely operate in different tissues. For example, a complex containing BRCA1 inhibits c-Myc-induced hTERT promoter activity in breast cancer cells (23), estrogen upregulates telomerase in both mammary and ovarian epithelial cells (3,29), and E2F-1 activates the hTERT promoter in some normal human somatic cells (44).The human papillomavirus type 16 (HPV16) E6 oncoprotein can induce telomerase activity in human primary keratinocytes through induction of hTERT expression (9,10,33,39). Studies on how E6 regulates hTERT have implicated E-box and X-box elements in the hTERT promoter (9, 10, 33, 40). c-Myc's role in directly enhancing transcription of hTERT has been demonstrated in several cell lines (15,21,45). However, the mechanism is still controversial and not well defined (9, 33, 39, 40). Although both E-box and c-Myc activities were found to be required for E6-mediated activation of hTERT (33,39,40), neither increased c-Myc protein level nor enhanced MycMax complex formation was detected with E6 expression (9,33,39,40).Recently, we identified NFX1-91 as a transcriptional repressor that binds to a consensus X-box element at the hTERT promoter, which overlaps with an E box (10). Two isoforms of NFX1 generated by alternative splicing...