BDNF and Hippocampal Synaptic Plasticity.

Brain-derived neurotrophic factor (BDNF) belongs to a family of small secreted proteins that also include nerve growth factor, neurotrophin 3, and neurotrophin 4. BDNF stands out among all neurotrophins by its high expression levels in the brain and its potent effects at synapses. Several aspects of BDNF biology such as transcription, processing, and secretion are regulated by synaptic activity. Such observations prompted the suggestion that BDNF may regulate activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP), a sustained enhancement of excitatory synaptic efficacy thought to underlie learning and memory. Here, we will review the evidence pointing to a fundamental role of this neurotrophin in LTP, especially within the hippocampus. Prominent questions in the field, including the release and action sites of BDNF during LTP, as well as the signaling and molecular mechanisms involved, will also be addressed. The diverse effects of BDNF at excitatory synapses are determined by the activation of TrkB receptors and downstream signaling pathways, and the functions, typically opposing in nature, of its immature form (proBDNF). The activation of p75NTR receptors by proBDNF and the implications for long-term depression will also be addressed. Finally, we discuss the synergy between TrkB and glucocorticoid receptor signaling to determine cellular responses to stress.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons.

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.

Ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous RIP3.

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis, a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally, we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels, with a concomitant increase of the RIP1 protein. Together, these two related proteins form the necrosome, the complex responsible for induction of necroptotic cell death. Interestingly, we found that caspase-8 mRNA, a known negative regulator of necroptosis, was transiently decreased following OGD. Importantly, we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia, specifically in the CA1 area of the hippocampus. Moreover, we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together, our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury.

Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level.

Proteomics-based technologies in the discovery of biomarkers for multiple sclerosis in the cerebrospinal fluid.

Multiple Sclerosis is the most common non-traumatic disorder of the central nervous system and is generally regarded as an immune-mediated disorder that occurs in young adults. Since cerebrospinal fluid is in close contact with the extracellular surface of the brain, it is of great interest to examine possible biomarkers for multiple sclerosis. Proteomic studies of cerebrospinal fluid samples represent an important step towards a better understanding of the disease and may lead to the identification of clinically useful markers. Methodological advances in proteomics allowed the comparison of the protein content in different cerebrospinal fluid samples, using gel or liquid-based approaches coupled with mass spectrometry. In this paper, we discuss the advantages and limitations of the strategies employed and the potential biomarkers for multiple sclerosis identified so far using proteomics-based approaches.

Excitotoxicity through Ca2+-permeable AMPA receptors requires Ca2+-dependent JNK activation.

The GluA4-containing Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (Ca-AMPARs) were previously shown to mediate excitotoxicity through mechanisms involving the activator protein-1 (AP-1), a c-Jun N-terminal kinase (JNK) substrate. To further investigate JNK involvement in excitotoxic pathways coupled to Ca-AMPARs we used HEK293 cells expressing GluA4-containing Ca-AMPARs (HEK-GluA4). Cell death induced by overstimulation of Ca-AMPARs was mediated, at least in part, by JNK. Importantly, JNK activation downstream of these receptors was dependent on the extracellular Ca(2+) concentration. In our quest for a molecular link between Ca-AMPARs and the JNK pathway we found that the JNK interacting protein-1 (JIP-1) interacts with the GluA4 subunit of AMPARs through the N-terminal domain. In vivo, the excitotoxin kainate promoted the association between GluA4 and JIP-1 in the rat hippocampus. Taken together, our results show that the JNK pathway is activated by Ca-AMPARs upon excitotoxic stimulation and suggest that JIP-1 may contribute to the propagation of the excitotoxic signal.

Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.

The use of preparations from Bryophyllum pinnatum in tocolysis is supported by both clinical (retrospective comparative studies) and experimental (using uterus strips) evidence. We studied here the effect of B. pinnatum juice on the response of cultured human myometrial cells to stimulation by oxytocin, a hormone known to be involved in the control of uterine contractions by increasing the intracellular free calcium concentration ([Ca2+]i). In this work, [Ca2+]i was measured online during stimulation of human myometrial cells (hTERT-C3 and M11) with oxytocin, which had been pre-incubated in the absence or in the presence of B. pinnatum juice. Since no functional voltage-gated Ca2+ channels could be detected in these myometrial cells, the effect of B. pinnatum juice was as well studied in SH-SY5Y neuroblastoma cells, which are known to have such channels and can be depolarised with KCl. B. pinnatum juice prevented the oxytocin-induced increase in [Ca2+]i in hTERT-C3 human myometrial cells in a dose-dependent manner, achieving a ca. 80% inhibition at a 2% concentration. Comparable results were obtained with M11 human primary myometrial cells. In hTERT-C3 cells, prevention of the oxytocin-induced increase in [Ca2+]i was independent of the extracellular Ca2+ concentration and of voltage-dependent Ca2+-channels. B. pinnatum juice delayed, but did not prevent the depolarization-induced increase in [Ca2+]i in SH-SY5Y cells. Taken together, the data suggest a specific and concentration-dependent effect of B. pinnatum juice on the oxytocin signalling pathway, which seems to corroborate its use in tocolysis. Such a specific mechanism would explain the rare and minor side-effects in tocolysis with B. pinnatum as well as its high therapeutic index.

Regulation of AMPA receptors and synaptic plasticity.

Neuronal activity controls the strength of excitatory synapses by mechanisms that include changes in the postsynaptic responses mediated by AMPA receptors. These receptors account for most fast responses at excitatory synapses of the CNS, and their activity is regulated by various signaling pathways which control the electrophysiological properties of AMPA receptors and their interaction with numerous intracellular regulatory proteins. AMPA receptor phosphorylation/dephosphorylation and interaction with other proteins control their recycling and localization to defined postsynaptic sites, thereby regulating the strength of the synapse. This review focuses on recent advances in the understanding of the molecular mechanisms of regulation of AMPA receptors, and the implications in synaptic plasticity.

Role of the brain-derived neurotrophic factor at glutamatergic synapses.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function, particularly of the glutamatergic synapses. BDNF may be released in the mature form, which activates preferentially TrkB receptors, or as proBDNF, which is coupled to the stimulation of the p75(NTR). In the mature form BDNF induces rapid effects on glutamate release, and may induce short- and long-term effects on the postsynaptic response to the neurotransmitter. BDNF may affect glutamate receptor activity by inducing the phosphorylation of the receptor subunits, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. Stimulation of the local protein synthesis and transcription activity account for the delayed effects of BDNF on glutamatergic synaptic strength. Several evidences show impaired synaptic plasticity of glutamatergic synapses in diseases where compromised BDNF function has been observed, such as Huntington's disease, depression, anxiety, and the BDNF polymorphism Val66Met, suggesting that upregulating BDNF-activated pathways may be therapeutically relevant. This review focuses on recent advances in the understanding of the regulation of the glutamatergic synapse by BDNF, and its implications in synaptic plasticity.

Effect of skin sensitizers on inducible nitric oxide synthase expression and nitric oxide production in skin dendritic cells: role of different immunosuppressive drugs.

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

Contact sensitizers downregulate the expression of the chemokine receptors CCR6 and CXCR4 in a skin dendritic cell line.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO(4)), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression.

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways.

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.

Dexamethasone-induced and estradiol-induced CREB activation and annexin 1 expression in CCRF-CEM lymphoblastic cells: evidence for the involvement of cAMP and p38 MAPK.

AIMS: Annexin 1 (ANXA1), a member of the annexin family of calcium-binding and phospholipid-binding proteins, is a key mediator of the anti-inflammatory actions of steroid hormones. We have previously demonstrated that, in the human lymphoblastic CCRF-CEM cell line, both the synthetic glucocorticoid hormone, dexamethasone (Dex), and the estrogen hormone, 17beta-estradiol (E2beta), induce the synthesis of ANXA1, by a mechanism independent of the activation of their nuclear receptors. Recently, it was reported that the gene coding for ANXA1 contains acAMP-responsive element (CRE). In this work, we investigated whether Dex and E2beta were able to induce the activation of CRE binding proteins (CREB) in the CCRF-CEM cells. Moreover, we studied the intracellular signalling pathways involved in CREB activation and ANXA1 synthesis in response to Dex and E2beta; namely, the role of cAMP and the p38 mitogen activated protein kinase (MAPK). RESULTS: The results show that Dex and E2beta were as effective as the cAMP analogue, dBcAMP, in inducing CREB activation. On the contrary, dBcAMP induced ANXA1 synthesis as effectively as these steroid hormones. Furthermore, the cAMP antagonist, Rp-8-Br-cAMPS, and the specific p38 MAPK inhibitor,SB203580, effectively prevented both Dex-induced, E2beta-induced and dBcAMP-induced CREB activation and ANXA1 synthesis. CONCLUSIONS: Taken together, our results suggest that,in CCRF-CEM cells, Dex-induced and E2beta-inducedANXA1 expression requires the activation of the transcription factor CREB, which in turn seems to be mediated by cAMP and the p38 MAPK. These findings also suggest that, besides the nuclear steroid hormone receptors, other transcription factors, namely CREB, may play important roles in mediating the anti-inflammatory actions of glucocorticoids and oestrogen hormones.

Dexamethasone prevents interleukin-1beta-induced nuclear factor-kappaB activation by upregulating IkappaB-alpha synthesis, in lymphoblastic cells.

AIMS: Glucocorticoids (GCs) exert some of their anti-inflammatory actions by preventing the activation of the transcription factor nuclear factor (NF)-kappaB. The GC-dependent inhibition of NF-kappaB may occur at different levels, but the mechanisms involved are still incompletely understood. In this work, we investigated whether the synthetic GC, dexamethasone (Dex), modulates the activity of NF-kappaB in the lymphoblastic CCRF-CEM cell line. We also evaluated the ability of Dex to prevent the activation of NF-kappaB in response to the potent proinflammatory cytokine, interleukin (IL)-1beta. RESULTS: Exposure of the cells to Dex (1 microM) induced the rapid degradation of IkappaB-alpha, leading to the transient translocation of the NF-kappaB family members p65 and p50 from the cytoplasm to the nucleus, as evaluated by western blot. Electrophoretic mobility shift assays revealed that, in the nucleus, these NF-kappaB proteins formed protein-DNA complexes, indicating a transient activation of NF-kappaB. Additionally, Dex also induced de novo synthesis of IkappaB-alpha, following its degradation. Finally, when the cells were exposed to Dex (1 microM) prior to stimulation with IL-1beta (20 ng/ml), Dex was efficient in preventing IL-1beta-induced NF-kappaB activation. The GC antagonist, RU 486 (10 microM), did not prevent any of the effects of Dex reported here. CONCLUSION: Our results indicate that, in CCRF-CEM cells, Dex prevents NF-kappaB activation, induced by IL-1beta, by a mechanism that involves the upregulation of IkappaB-alpha synthesis, and that depends on the early and transient activation of NF-kappaB.

Dexamethasone induces the secretion of annexin I in immature lymphoblastic cells by a calcium-dependent mechanism.

The mechanisms by which glucocorticoids (GC) regulate annexin I (ANXA1) secretion in different cells are still a matter of debate. The aims of this study were to evaluate the ability of dexamethasone (Dex) to induce ANXA1 secretion and to investigate the roles of the intracellular free Ca2+ concentration ([Ca2+]i), and of the GC receptor, on that process. For this purpose, the human immature lymphoblastic CCRF-CEM cell line was used. Treatment of the cells with Dex, for up to 4 h, significantly reduced the intracellular content of ANXA1 and increased the amount of this protein bound to the outer surface of the plasma membrane, whereas exposure of cells to Dex, for 12 h, induced the synthesis of ANXA1. At the same short time periods, Dex also induced a significant increase in the [Ca2+]i. Incubation of the cells with BAPTA-AM (10 microM), a cell-permeant high affinity Ca2+ chelator, completely inhibited Dex-induced ANXA1 secretion. Furthermore, the Ca2+ ionophore, ionomycin, alone induced ANXA1 cleavage, but not its secretion. Additionally, we used brefeldin A to investigate the involvement of the classical endoplasmic reticulum (ER)-Golgi pathway of protein secretion in the release of ANXA1. The GC receptor antagonist, RU486, neither reverted the Dex-dependent ANXA1 secretion nor inhibited the increase of the [Ca2+]i induced by Dex. Together, our results indicate that Dex induces ANXA1 synthesis and secretion in CCRF-CEM cells. ANXA1 secretion in this cell type show the following characteristics: (i) is unlikely to involve the classical ER-Golgi pathway; (ii) requires a Ca(2+)-dependent cleavage of ANXA1; (iii) involves both Ca(2+)-dependent and independent mechanisms; and (iv) is apparently independent of the GC receptor alpha isoform.

Non-specific effects of the MEK inhibitors PD098,059 and U0126 on glutamate release from hippocampal synaptosomes.

In order to investigate a role for the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) on hippocampal neurotransmitter release, we studied the effect of commonly used MEK (mitogen-activated protein kinase [MAPK]/ERK kinase) inhibitors, PD098,059 and U0126, on depolarization-induced glutamate release. PD098,059 inhibited glutamate release from hippocampal synaptosomes stimulated with 15 mM KCl in a concentration-dependent manner. At the same range of concentrations, PD098,059 inhibited basal and KCl-stimulated ERK1/2 phosphorylation. U0126, however, did not significantly affect KCl-evoked glutamate release at concentrations shown to inhibit ERK activity. Nonetheless, U0126 unspecifically potentiated depolarization-induced Ca2+-independent glutamate release, which masked a small dose-dependent inhibitory effect on the Ca2+-dependent release. PD098,059 reduced the [Ca2+]i response to KCl by partially inhibiting Ca2+ entry through N- and P-/Q-type voltage-gated Ca2+ channels, whereas U0126 did not affect depolarization-induced Ca2+ influx. To overcome the unspecific effect of PD098,059 on Ca2+ entry, we studied the effect of both MEK inhibitors on glutamate release stimulated by a Ca2+ ionophore. PD098,029 and U0126 showed a small dose-dependent inhibitory effect on ionomycin-induced glutamate release, at concentrations shown to inhibit ionomycin-stimulated ERK phosphorylation. These findings uncover new unspecific actions for both MEK inhibitors and suggest a minor role for ERK in modulating glutamate release in the hippocampus.

LPS induction of I kappa B-alpha degradation and iNOS expression in a skin dendritic cell line is prevented by the janus kinase 2 inhibitor, Tyrphostin b42.

The Janus kinase (JAK) family of protein tyrosine kinases are activated in response to a wide variety of external stimuli. Here we have investigated whether the janus kinase 2 (JAK2) is involved in the induction of nitric oxide synthase type II (iNOS) expression in a mouse fetal skin dendritic cell line (FSDC). In FSDC the expression of iNOS protein and nitric oxide production, in response to the lipopolysaccharide (LPS) stimulus (5 microg/ml), is inhibited by the specific inhibitor of the JAK2, tyrphostin B42 with an half maximal inhibitory concentration (IC(50)) of 9.65 microM. The antioxidant compound pyrrolidinedithiocarbamate (PDTC) inhibits both the nitrite production with an IC(50) of 16.6 microM and the iNOS protein expression in FSDC. In addition, LPS induces the activation of NF-kappa B, and tyrphostin B42 prevents the degradation of the cytosolic factor I kappa B-alpha and blocks the translocation of the NF-kappa B p65 protein subunit into the nucleus. These results indicate that the JAK family of protein kinases and the transcription factor NF-kappa B are involved in the induction of iNOS protein expression in FSDC stimulated with LPS.

Granulocyte-macrophage colony-stimulating factor activates the transcription of nuclear factor kappa B and induces the expression of nitric oxide synthase in a skin dendritic cell line.

Nitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM-CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal-skin-derived dendritic cell line (FSDC) and, consequently, increases the nitrite production from 11.9 +/- 3.2 micromol/L (basal level) to 26.9 +/- 4.2 micromol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC50) of 19.3 micromol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM-CSF induces the phosphorylation and degradation of the inhibitor of NF-kappaB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF-kappaB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM-CSF activates the transcription factor NF-kappaB. Together, these results show that GM-CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF-kappaB pathway.