Participation of protease-activated receptor-1 in thrombin-induced microglial activation

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-alpha (TNF-alpha) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-alpha production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.     

Divergent effect of proteasome inhibition on interleukin-1beta and tumor necrosis factor alpha signaling in human astroglial cells

Impaired functioning of the proteasome pathway is one of the molecular mechanism underlying neurodegenerative changes in Alzheimer's disease. In this study, we report that dysfunction of the proteasome pathway in astroglial cells leads to decreased survival and dysregulation of chemokines by differential regulation of the nuclear factor kappa B and c-jun N-terminal kinase (JNK) pathways. We further demonstrated that proteasome inhibition augmented interleukin-1 beta- and tumor necrosis factor-alpha-induced activation of the IkappaBalpha kinase and MKK4/JNK/c-Jun pathway along with TAK1 activation. These results suggest that impaired function of the proteasome pathway may potentiate the immuno-pathologic role of secondarily activated astrocytes in the brain.     

Evidence for functional adenosine A3 receptors in microglia cells

Adenosine exerts its effects through four subtypes of G-protein-coupled receptors (GPCRs): adenosine A1 and A3 receptors (A3R), which generally couple to Gi proteins and adenosine A2A and A2B receptors that activate Gs proteins. Though there is evidence for the expression of mRNA for the A3R in the central nervous system, evidence for functional receptors has depended on drugs with uncertain specificity. Here, we show that A3Rs mediating functional responses are present in microglia cells. By selectively stimulating the A3R in both primary mouse microglia cells and the N13 microglia cell line with the agonist Cl-IB-MECA, we have found a biphasic, partly Gi protein-dependent influence on the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2). ERK1/2 activation was assessed by immunoblotting with phospho-specific antibodies. The involvement of the A3R in Cl-IB-MECA-induced ERK1/2 phosphorylation was confirmed by demonstrating that those effects are absent in primary mouse microglia cells isolated from mice lacking the gene for the A3R.     

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

17beta-oestradiol up-regulates longevity-related, antioxidant enzyme expression via the ERK1 and ERK2[MAPK]/NFkappaB cascade

Females live longer than males. Oestrogens protect females against aging by up-regulating the expression of antioxidant, longevity-related genes such as glutathione peroxidase (GPx) and Mn-superoxide dismutase (Mn-SOD). The mechanism through which oestrogens up-regulate those enzymes remains unidentified, but may have implications for gender differences in lifespan. We show that physiological concentrations of oestradiol act through oestrogen receptors to reduce peroxide levels in MCF-7 cells (a mammary gland tumour cell line). Oestradiol increases MAP kinase (MAPK) activation as indicated by ERK1 and ERK2 phosphorylation in MCF-7 cells, which in turn activates the nuclear factor kappa B (NFkappaB) signalling pathways as indicated by an increase in the p50 subunit of NFkappaB in nuclear extracts. Blockade of MAPK and NFkappaB signalling reduces the antioxidant effect of oestradiol. Finally, we show that activation of MAPK and NFkappaB by oestrogens drives the expression of the antioxidant enzymes Mn-SOD and GPx. We conclude that oestradiol sequentially activates MAPK and NFkappaB following receptor activation to up-regulate the expression of antioxidant enzymes, providing a cogent explanation for the antioxidant properties of oestrogen and its effects on longevity-related genes.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.     

Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells

The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation.     

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P(1) and S1P(3), as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P(1)/S1P(3) receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT.     

Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D3-II: calcium transport, receptor kinetics, and signal transduction in intestine of female chickens

We recently reported (Larsson and Nemere [2003]: Endocrinology 144:1726) the effects of growth and maturation on 1,25(OH)2D3-membrane initiated effects in the intestine of male chickens. Here we extend our observations to studies on females with two stages of high calcium demand: growth (7-14 weeks) and egg laying (28-58 weeks). The rapid stimulatory effect of 130 pM 1,25(OH)2D3 on calcium transport was assessed as a physiological response in perfused duodena of 7-, 14-, 28-, and 58-week-old chickens, and determined to be 308%, 184%, 170%, and 153%, respectively, of corresponding controls after 40 min. Saturation analyses of [3H]1,25(OH)2D3 binding to nuclear vitamin D receptor (VDR) indicated an absence of cooperative binding, no changes in dissociation constant (Kd) with age, and an increase in maximum binding capacity (Bmax) between 7-week birds and older age groups. Analyses of saturable binding of [3H]1,25(OH)2D3 to the membrane associated rapid response steroid binding protein (1,25D3-MARRS bp) in basal lateral membranes (BLM), indicated cooperative binding, and an increase in both Bmax and Kd with age. No changes in the age-related expression of 1,25D3-MARRS bp were found, as judged by Western analyses, suggesting that a shift in ligand binding to lower affinity membrane components accounted for the increase in calculated Bmax. Basal levels of protein kinase C (PKC) activity decreased with age, as did hormone enhancement of activity. Basal levels of protein kinase A (PKA) activity remained constant with age, while the magnitude of hormone stimulation increased. Comparison of dose-response curves for ion transport and kinase activities in 7-week chicks suggested that PKC mediates phosphate transport while PKA mediates calcium transport. Thus, the age-related loss of calcium transport is most likely related to loss of PKC-mediated phosphate transport.