Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp^−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp^−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp^−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.     

Role of hydroxyl containing residues in the intracellular region of rat bradykinin B(2) receptor in signal transduction, receptor internalization, and resensitization

In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.     

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22)

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

Opioid Control of the In Vitro Release of Cholecystokinin-Like Material from the Rat Substantia Nigra

Possible interactions between Met-enkephalin and cholecystokinin (CCK)-containing neurons in the rat substantia nigra were investigated by looking for the effects of various opioid receptor ligands and inhibitors of enkephalin-degrading enzymes on the K(+)-evoked overflow of CCK-like material (CCKLM) from substantia nigra slices. The delta-opioid agonists D-Pen2, D-Pen5-enkephalin (50 microM) and Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET; 3 microM) enhanced, whereas the mu-opioid agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and MePhe3, D-Pro4-morphiceptin (PL 017; 10 microM) decreased, the K(+)-evoked release of CCKLM. By contrast, the kappa-opioid agonist U-50488 H (5 microM) was inactive. The stimulatory effect of DTLET could be prevented by the delta antagonist ICI-154129 (50 microM), but not by the mu antagonist naloxone (1 microM). Conversely, the latter drug, but not ICI-154129, prevented the inhibitory effect of DAGO and PL 017. A significant increase in CCKLM overflow was observed upon tissue superfusion with the peptidase inhibitors kelatorphan or bestatin plus thiorphan. This effect probably resulted from the stimulation of delta-opioid receptors by endogenous enkephalins protected from degradation, because it could be prevented by ICI-154129 (50 microM). Furthermore the peptidase inhibitors did not enhance CCKLM release further when delta-opioid receptors were stimulated directly by DTLET (3 microM). These data indicate that opioids acting on delta and mu receptors may exert an opposite influence, i.e., excitatory and inhibitory, respectively, on CCK-containing neurons in the rat substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)