Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide

Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-kappaB DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.     

Molecular mechanisms involved in the actions of apotransferrin upon the central nervous system: Role of the cytoskeleton and of second messengers

Apotransferrin (aTf), intracranially administered into newborn rats, produces increased myelination with marked increases in the levels of myelin basic protein (MBP), phospholipids and galactolipids, and mRNAs of MBP and 2', 3' cyclic nucleotide 3'-phosphohydrolase (CNPase). Cytoskeletal proteins such as tubulin, actin, and microtubule-associated proteins are also increased after aTf injection. In contrast, almost no changes are observed in myelin proteolipid protein (PLP) or in its mRNA or cholesterol. In the present study, we used brain-tissue slices and cell cultures highly enriched for oligodendroglia to investigate signaling pathways involved in the action of aTf, and to find out whether cytoskeletal integrity and dynamics were essential for its action upon the neural expression of certain genes. Treatment of brain-tissue slices with aTf produced a marked increase in the expression of MBP, CNPase, and tubulin mRNAs. Colchicine, cytochalasin, and taxol severely reduced the effect of aTf. Addition to cultures of an antibody against transferrin receptor (TfR), protein kinase inhibitors, or a cyclic AMP (cAMP) analogue showed that a functionally intact TfR was necessary, and that tyrosine kinase, protein kinase C and A, as well as calcium-calmodulin-dependent kinase (Ca-CaMK) activities appeared to mediate aTf actions upon the expression of the above mentioned genes. Changes in the levels of phosphoinositides and cAMP induced by aTf in oligodendroglial cell (OLGc) cultures correlated with these results and coincide with an activation of the cyclic response element binding protein (CREB) and of mitogen activated protein kinases. The increased expression of certain myelin genes produced by aTf appear to be mediated by interaction of this glycoprotein with its receptor, by the cytoskeleton of the OLGc, and by a complex activation of protein kinases which lead to CREB phosphorylation.     

Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine

We recently showed that Nexrutine^R, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our data also indicate that the anti-proliferative effects of Nexrutine^R are emediated in part by Akt and Cyclic AMP response element binding protein (CREB).Cyclooxygenase (Cox-2), a pro-inflammatory mediator, is a CREB target that induces prostaglandin E₂ (PGE₂) and suppresses apoptosis. Treatment of LNCaP cells with Nexrutine^R reduced tumor necrosis factor α-induced enzymatic as well as promoter activities of Cox-2. Nexrutine^R also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating Nexrutine^R response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01). We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like Nexrutine^R, demonstrating a prospective for development of Nexrutine^R for prostate cancer management.     

Effect of cyclic AMP on the expression of myelin basic protein species and myelin proteolipid protein in committed oligodendrocytes: differential involvement of the transcription factor CREB

Our previous results support the idea that CREB (cyclic AMP-response element binding protein) may be a mediator of neuroligand and growth factor signals that, coupled to different signal transduction pathways, play different roles at specific stages of oligodendrocyte development. In the early stages, when cells are immature precursors, CREB may play a role as a mediator of protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) pathways regulating cell proliferation. In contrast, at a later stage, when cells are already committed oligodendrocytes, CREB seems to play an important role as a mediator in the stimulation of myelin basic protein (MBP) expression by cyclic AMP (cAMP). In this study, we have investigated whether cAMP and CREB play a role in regulating the expression of all or on the other hand particular MBP isoforms. The results indicated that treatment of committed oligodendrocytes with the cAMP analogue db-cAMP results in a pattern of expression of MBP-related polypeptides that most closely resembles the pattern of MBPs observed in cerebra from adult animals. Experiments in which CREB expression was inhibited using a CREB antisense oligonucleotide, suggested that CREB is involved in the cAMP-dependent stimulation of all the MBP isoforms. In contrast, we have found that db-cAMP stimulates the expression of myelin proteolipid protein (PLP) in a process that occurs despite inhibition of CREB expression. These results support the idea that cAMP stimulates the maturation of oligodendrocytes and stress the fact multiple mechanisms may convey the action of this second messenger modulating oligodendrocyte differentiation and myelination.     

DARPP-32 and CREB are present in type 2 iodothyronine deiodinase-producing tanycytes: implications for the regulation of type 2 deiodinase activity

Type 2 iodothyronine deiodinase, an enzyme involved in the conversion of thyroxin to the biologically active 3,5, 3'-triiodothyronine, is highly concentrated in a group of specialized ependymal cells, tanycytes, lining the wall and floor of the third ventricle. As this distribution is highly reminiscent of the distribution of cells containing the phosphatase inhibitor, DARPP-32, we raised the possibility that these two proteins may coexist in tanycytes and that DARPP-32 may modulate type 2 deiodinase activity by regulating the phosphorylation state of the cAMP regulatory factor, CREB. To address this question, double-labeling histochemical studies were performed for type 2 deiodinase mRNA and DARPP-32 immunoreactivity (IR), or DARPP-32- and CREB-IR in the same tissue sections. Type 2 deiodinase mRNA was found in the cell bodies of all DARPP-32-immunolabeled tanycytes. Both type 2 deiodinase mRNA and DARPP-32-IR also extended into tanycyte processes that ramified in the arcuate nucleus and median eminence, in close association with blood vessels and portal capillaries. In contrast, type 2 deiodinase mRNA was not present in the same cells that contained DARPP-32-IR in the pituitary gland. All tanycytes containing DARPP-32-IR also contained CREB-IR in their nucleus. Since type 2 deiodinase activity can be induced by substances that increase cAMP, we hypothesize that DARPP-32 may regulate the activity of type 2 deiodinase by prolonging the activation of CREB. Selectivity for the colocalization of these factors to tanycytes but not the pituitary gland, may explain the heterogeneous response of type 2 deiodinase activity in these two loci in response to specific stimuli such as fasting.     

桂枝汤对高、低体温大鼠下丘脑组织转录因子CREB的影响

目的：探讨体温变化时下丘脑组织中磷酸化cAMP反应元件结合蛋白（pCREB）的活性及桂枝汤调节体温作用与此关系。方法：选用前列腺素受体3（EP3）发热大鼠模型及氨基比林低体温大鼠模型，采用比色法测定给予桂枝汤后大鼠下丘脑组织中pCREB活性。结果：两种模型下丘脑组织中pCREB活性均有升高倾向，以发热大鼠明显；给予桂枝汤可使发热大鼠的pCREB活性降低，而低体温大鼠的pCREB活性变化不明显。结论：pCREB参与了大鼠的体温变化过程；桂枝汤的解热作用可能与降低pCREB活性有关；pCREB基本上不参与低体温机制的调节或仅有较弱调节作用。桂枝汤的升温作用与其关系不明显。