To further our understanding of the structure and function of phosphodiesterases of the newly identified family of high affinity cAMP-specific phosphodiesterases (PDE7), we identified and characterized the isozyme expressed in human skeletal muscle and the protein product of the previously isolated isozyme HCP1 (designated HSPDE7A1). We report the isolation of a cDNA encoding the full-length skeletal muscle isoform of human PDE7A (HSPDE7A2). The DNA sequence of this skeletal muscle cDNA indicates that PDE7A2 is a novel 5 splice variant of PDE7A encoding an isoform with a novel, hydrophobic N terminus. The 456-amino acid PDE7A2 protein is detected on Western blots as a band with an apparent mobility of 50 kDa. PDE7A2 is a high affinity cAMP-specific PDE (K m ‫؍‬ 0.1 M), which is localized to particulate cellular fractions. The PDE7A1 (HCP1) isozyme is detected on Western blots as a band with an apparent mobility of 57 kDa, demonstrating that the previously isolated HCP1 cDNA encodes the fulllength PDE7A1 protein. The even distribution of PDE7A mRNA among fetal tissues and the relative abundance of its two mRNAs strongly suggest that the expression of PDE7A is regulated throughout development.

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The management of neuropathic pain is still a major challenge because of its unresponsiveness to most common treatments. Curcumin has been reported to play an active role in the treatment of various neurological disorders, such as neuropathic pain. Curcumin has long been recognized as a p300/CREB-binding protein (CBP) inhibitor of histone acetyltransferase (HAT) activity. However, this mechanism has never been investigated for the treatment of neuropathic pain with curcumin. The aim of the present study was to investigate the anti-nociceptive role of curcumin in the chronic constriction injury (CCI) rat model of neuropathic pain. Furthermore, with this model we investigated the effect of curcumin on P300/CBP HAT activity-regulated release of the pro-nociceptive molecules, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (Cox-2). Treatment with 40 and 60 mg/kg body weight curcumin for 7 consecutive days significantly attenuated CCI-induced thermal hyperalgesia and mechanical allodynia, whereas 20 mg/kg curcumin showed no significant analgesic effect. Chromatin immunoprecipitation analysis revealed that curcumin dose-dependently reduced the recruitment of p300/CBP and acetyl-Histone H3/acetyl-Histone H4 to the promoter of BDNF and Cox-2 genes. A similar dose-dependent decrease of BDNF and Cox-2 in the spinal cord was also observed after curcumin treatment. These results indicated that curcumin exerted a therapeutic role in neuropathic pain by down-regulating p300/CBP HAT activity-mediated gene expression of BDNF and Cox-2.

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Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes. The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.

Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd−/− compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.

BackgroundNeuropathic pain is detrimental to human health; however, its pathogenesis still remains largely unknown. Overexpression of pain-associated genes and increased nociceptive somato-sensitivity are well observed in neuropathic pain. The importance of epigenetic mechanisms in regulating the expression of pro- or anti-nociceptive genes has been revealed by studies recently, and we hypothesize that the transcriptional coactivator and the histone acetyltransferase E1A binding protein p300 (p300), as a part of the epigenetic mechanisms of gene regulation, may be involved in the pathogenesis of neuropathic pain induced by chronic constriction injury (CCI). To test this hypothesis, two different approaches were used in this study: (I) down-regulating p300 with specific small hairpin RNA (shRNA) and (II) chemical inhibition of p300 acetyltransferase activity by a small molecule inhibitor, C646.ResultsUsing the CCI rat model, we found that the p300 expression was increased in the lumbar spinal cord on day 14 after CCI. The treatment with intrathecal p300 shRNA reversed CCI-induced mechanical allodynia and thermal hyperalgesia, and suppressed the expression of cyclooxygenase-2 (COX-2), a neuropathic pain-associated factor. Furthermore, C646, an inhibitor of p300 acetyltransferase, also attenuated mechanical allodynia and thermal hyperalgesia, accompanied by a suppressed COX-2 expression, in the spinal cord.ConclusionsThe results suggest that, through its acetyltransferase activity in the spinal cord after CCI, p300 epigenetically plays an important role in neuropathic pain. Inhibiting p300, using interfering RNA or C646, may be a promising approach to the development of new neuropathic pain therapies.