Requirement of extracellular signal-regulated kinase/mitogen-activated protein kinase for long-term potentiation in adult mouse anterior cingulate cortex

Long-term potentiation (LTP) in the anterior cingulate cortex (ACC) is believed to be critical for higher brain functions including emotion, learning, memory and chronic pain. N-methyl-D-aspartate (NMDA) receptor-dependent LTP is well studied and is thought to be important for learning and memory in mammalian brains. As the downstream target of NMDA receptors, the extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade has been extensively studied for its involvement in synaptic plasticity, learning and memory in hippocampus. By contrast, the role of ERK in cingulate LTP has not been investigated. In this study, we examined whether LTP in ACC requires the activation of ERK. We found that P42/P44 MAPK inhibitors, PD98059 and U0126, suppressed the induction of cingulate LTP that was induced by presynaptic stimulation with postsynaptic depolarization (the pairing protocol). We also showed that cingulate LTP induced by two other different protocols was also blocked by PD98059. Moreover, we found that these two inhibitors had no effect on the maintenance of cingulate LTP. Inhibitors of c-Jun N-terminal kinase (JNK) and p38, other members of MAPK family, SP600125 and SB203850, suppressed the induction of cingulate LTP generated by the pairing protocol. Thus, our study suggests that the MAPK signaling pathway is involved in the induction of cingulate LTP and plays a critical role in physiological conditions.     

A requirement of nuclear factor-kappaB activation in fear-potentiated startle

Previous studies have shown that biochemical changes that occur in the amygdala during fear conditioning in vivo are similar to those occur during long term potentiation (LTP) in vitro. Electrophoretic mobility shift assay of nuclear extracts from startle-potentiated rats showed a selective increase in the amygdala of nuclear factor-kappaB (NF-kappaB) DNA binding activity. Supershift experiments further indicated that p65 and p50 subunits but not c-Rel were involved in DNA binding. The protein levels of IkappaB-alpha were reduced by treatments that reliably induced LTP in this area of the brain. This was accompanied by a decrease of NF-kappaB in the cytoplasm concomitant with an increase in the nucleus. Quantitative analysis of IkappaB kinase activity demonstrated that fear training led to an increase in kinase activity, and this effect was inhibited by thalidomide. Paralleled behavioral tests revealed that thalidomide inhibited fear-potentiated startle. Intra-amygdala administration of kappaB decoy DNA prior to training impaired fear-potentiated startle as well as LTP induction. Similarly, NF-kappaB inhibitors blocked IkappaB-alpha degradation and startle response. These results provide the first evidence of a requirement of NF-kappaB activation in the amygdala for consolidation of fear memory.     

Nerve growth factor activates brain-derived neurotrophic factor promoter IV via extracellular signal-regulated protein kinase 1/2 in PC12 cells

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.     

Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase

Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.     

Positive regulation of phagocytosis by SIRPbeta and its signaling mechanism in macrophages

SIRPbeta (signal-regulatory protein beta) is a transmembrane protein that is expressed in hematopoietic cells but whose functions are unknown. We have now cloned mouse SIRPbeta cDNA and have shown that the gene is expressed in various tissues in addition to cells of the macrophage lineage. Engagement of SIRPbeta by specific monoclonal antibodies promoted Fcgamma receptor-dependent or -independent phagocytosis in mouse peritoneal macrophages. It also induced marked activation of MAPK and the upstream kinase MEK but weak activation of Akt. MEK inhibitors markedly blocked the promotion of phagocytosis by SIRPbeta, whereas an inhibitor of phosphoinositide 3-kinase partly blocked such response. In addition, inhibitors of myosin light chain kinase or of myosin ATPase blocked the promotion of phagocytosis by SIRPbeta. Furthermore, SIRPbeta induced the formation of filopodia and lamellipodia in macrophages as well as the translocation of activated MAPK to these structures. It also elicited tyrosine phosphorylation of DAP12, Syk, and SLP-76, and a Syk inhibitor blocked the promotion of phagocytosis and activation of MAPK by SIRPbeta. Our results suggest that engagement of SIRPbeta promotes phagocytosis in macrophages by inducing the tyrosine phosphorylation of DAP12, Syk, and SLP-76 and the subsequent activation of a MEK-MAPK-myosin light chain kinase cascade.     

Persistent protein kinase activation in the maintenance phase of long-term potentiation.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a robust form of synaptic plasticity that may contribute to mammalian memory formation. A variety of pharmacological evidence suggests that persistent kinase activation contributes to the maintenance of LTP. To determine whether persistent activation of protein kinases was associated with the maintenance phase of LTP, protein kinase activity was measured in control and LTP samples using exogenous protein kinase substrates in an in vitro assay of homogenates of the CA1 region of rat hippocampal slices. After LTP, protein kinase activity was persistently increased, and the induction of this effect was blocked by the N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid. The increased protein kinase activity was found to be significantly attenuated by PKC(19-36), a selective peptide inhibitor of protein kinase C. Thus, LTP is associated with an N-methyl-D-aspartate receptor-mediated generation of a persistently activated form of protein kinase C. These data lend strong support to the model that persistent protein kinase activation contributes to the maintenance of LTP.     

Cell Adhesion Induces the Plasminogen Activator Inhibitor-1 Gene Expression through Phosphatidylinositol 3-Kinase/Akt Activation in Anchorage Dependent Cells

Type-I plasminogen activator inhibitor (PAI-1) is the primary inhibitor of both tissue- and urokinase-type plasminogen activators (t-PA, u-PA) and is thus a primary regulator of plasminogen activation and possibly of extracellular proteolysis. In anchorage-dependent cells, the PAI-1 gene was regulated by cell adhesion. PAI-1 gene expression was induced more evidently in cells adhered to the culture plate than in nonadherent cells. In this study, we investigated the signal pathway of the PAI-1 gene expression regulated by cell adhesion. We found the induction of both PAI-1 mRNA and protein, when cells adhered to culture dish, was inhibited by the PI-3 kinase specific inhibitors (Ly294002 and wortmannin). The cells seeded on collagen-1 coated plate with low serum further demonstrated that the PAI-1 gene expression was prolonged by the cell adhesion. The above-mentioned PI-3 kinase specific inhibitors also blocked the PAI-1 maintenance when cell adhered to collagen-1 coated plate. In addition, we found that both PI-3 kinase and its downstream molecule, Akt, were activated more evidently in adherent cells than in nonadherent cells. Furthermore, we transfected antisense oligodeoxynucleotides of Akt (AS-ODN-Akt) into cells to block the expression of Akt and found that the induction of PAI-1 mRNA was also inhibited. Hence, we conclude that the induction of PAI-1 gene expression is cell adhesion dependent and is through PI-3 kinase and Akt activation.     

Cell adhesion induces the plasminogen activator inhibitor-1 gene expression through phosphatidylinositol 3-kinase/Akt activation in anchorage dependent cells

Type-I plasminogen activator inhibitor (PAI-1) is the primary inhibitor of both tissue- and urokinase-type plasminogen activators (t-PA, u-PA) and is thus a primary regulator of plasminogen activation and possibly of extracellular proteolysis. In anchorage-dependent cells, the PAI-1 gene was regulated by cell adhesion. PAI-1 gene expression was induced more evidently in cells adhered to the culture plate than in nonadherent cells. In this study, we investigated the signal pathway of the PAI-1 gene expression regulated by cell adhesion. We found the induction of both PAI-1 mRNA and protein, when cells adhered to culture dish, was inhibited by the PI-3 kinase specific inhibitors (Ly294002 and wortmannin). The cells seeded on collagen-1 coated plate with low serum further demonstrated that the PAI-1 gene expression was prolonged by the cell adhesion. The above-mentioned PI-3 kinase specific inhibitors also blocked the PAI-1 maintenance when cell adhered to collagen-1 coated plate. In addition, we found that both PI-3 kinase and its downstream molecule, Akt, were activated more evidently in adherent cells than in nonadherent cells. Furthermore, we transfected antisense oligodeoxynucleotides of Akt (AS-ODN-Akt) into cells to block the expression of Akt and found that the induction of PAI-1 mRNA was also inhibited. Hence, we conclude that the induction of PAI-1 gene expression is cell adhesion dependent and is through PI-3 kinase and Akt activation.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells

The mechanisms for the effect of hyperglycemia on insulin-induced mitogenesis were investigated using rat vascular smooth muscle cells (VSMC). VSMC were preincubated in serum-free medium with low (5 mM) glucose (LG condition) or high (25 mM) glucose (HG condition), and examined for DNA synthesis using bromodeoxyuridine (BrdUrd) incorporation. Mitogen-activated protein kinase (MAPK) activity and MAPK phosphatase (MKP-1) protein expression were detected by Western blot analysis. Phosphatidylinositol 3-kinase (PI-3K) activity was detected by thin layer chromatography. Insulin induced a dose-dependent increase in BrdUrd incorporation (123.3+/-2.6% over basal level with 1 microM insulin) in the LG group and this effect was significantly enhanced (161.6+/-10.4% over basal level) in the HG group. In the LG group, MAPK activity was transient with a peak activation (137.4+/-11.2% over basal level) after 10 min exposure to 100 nM insulin. In the HG group, the MAPK activity was significantly potentiated (two-fold compared to the LG group) and was sustained even after 60 min. Insulin also induced PI-3K activity and MKP-1 expression, both of which were blocked by the PI-3K inhibitor wortmannin. In the HG group, insulin-induced PI-3K and MKP-1 expression was almost abolished. In conclusion, high glucose enhances insulin-induced mitogenesis associated with the potentiation of insulin-stimulated MAPK activity in VSMC. These effects of glucose might in part be due to the attenuation of MKP-1 expression through the blockage of the insulin-PI-3K signal pathway.     

Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1

In human neutrophils, IL-8 induces chemotaxis, the respiratory burst, and granule release, and enhances cellular adhesion, a beta(2) integrin-dependent event. IL-8 stimulates neutrophil adhesion to purified fibrinogen in a Mac-1-dependent manner. Mitogen-activated protein kinase (MAPK) activation was detected in human neutrophil lysates after treatment with IL-8 and PMA, but not the activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to fibrinogen was blocked 50% by the MAPK/extracellular signal-related kinase-activating enzyme inhibitor PD098059. Adhesion was blocked approximately 75% by inhibition of the phosphatidylinositol-3 kinase (PI3K) pathway with LY294002, supporting that activation of both MAPK and PI3K may play a role in IL-8-dependent inside-out signals that activate Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the presence of PI3K inhibitors LY294002 or wortmannin, supporting a model in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A 74-kDa molecular mass species was detected by an activation-specific Ab to PKC when cells were stimulated with PMA or IL-8, but not a beta(2)-activating Ab. Inhibition of either MAPK or PKC resulted in partial inhibition of IL-8-stimulated polymorphonuclear neutrophil adhesion, and treatment with both inhibitors simultaneously completely abolished IL-8-stimulated adhesion to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC activation, suggesting a branch point that precedes PI3K activation. These data suggest that both MAPK and PKC are activated in response to IL-8 stimulation, and that these may represent independent pathways for beta(2) integrin activation in neutrophils.     

Bradykinin B2 Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.     

Synergistic activation of dopamine D1 and TrkB receptors mediate gain control of synaptic plasticity in the basolateral amygdala

Fear memory formation is thought to require dopamine, brain-derived neurotrophic factor (BDNF) and zinc release in the basolateral amygdala (BLA), as well as the induction of long term potentiation (LTP) in BLA principal neurons. However, no study to date has shown any relationship between these processes in the BLA. Here, we have used in vitro whole-cell patch clamp recording from BLA principal neurons to investigate how dopamine, BDNF, and zinc release may interact to modulate the LTP induction in the BLA. LTP was induced by either theta burst stimulation (TBS) protocol or spaced 5 times high frequency stimulation (5xHFS). Significantly, both TBS and 5xHFS induced LTP was fully blocked by the dopamine D1 receptor antagonist, SCH23390. LTP induction was also blocked by the BDNF scavenger, TrkB-FC, the zinc chelator, DETC, as well as by an inhibitor of matrix metalloproteinases (MMPs), gallardin. Conversely, prior application of the dopamine reuptake inhibitor, GBR12783, or the D1 receptor agonist, SKF39393, induced robust and stable LTP in response to a sub-threshold HFS protocol (2xHFS), which does not normally induce LTP. Similarly, prior activation of TrkB receptors with either a TrkB receptor agonist, or BDNF, also reduced the threshold for LTP-induction, an effect that was blocked by the MEK inhibitor, but not by zinc chelation. Intriguingly, the TrkB receptor agonist-induced reduction of LTP threshold was fully blocked by prior application of SCH23390, and the reduction of LTP threshold induced by GBR12783 was blocked by prior application of TrkB-FC. Together, our results suggest a cellular mechanism whereby the threshold for LTP induction in BLA principal neurons is critically dependent on the level of dopamine in the extracellular milieu and the synergistic activation of postsynaptic D1 and TrkB receptors. Moreover, activation of TrkB receptors appears to be dependent on concurrent release of zinc and activation of MMPs.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

The neuropeptide pituitary adenylate cyclase activating protein is a physiological activator of human monocytes

Pituitary adenylate cyclase activating protein (PACAP) and its structurally related vasointestinal peptide (VIP) bind to three G-protein-coupled receptors named VPAC1 and VPAC2 for VIP/PACAP receptors and PAC1 for PACAP preferred receptors. We report that in freshly isolated human monocytes PACAP acts as a pro-inflammatory molecule. By RT-PCR, VPAC1 mRNA was the only receptor found to be expressed; VPAC1 protein was detected by Western blotting and visualized by immunohistochemistry. Signaling pathways activated by PACAP include the extracellular regulated kinase (ERK), the stress-activated MAPK p38, the focal adhesion kinase, Pyk2 and its associated cytoskeleton protein paxillin and the phosphatidylinositol 3-kinase (PI-3K). PACAP induces a transient peak in cytoplasmic calcium associated with an increase in reactive oxygen species production and upregulation in membrane expression of the integrin CD11b as well as the complement receptor 1. Control of the different pathways and functions stimulated by PACAP were evaluated using Phospholipase C (PLC), PI-3K, ERK and p38 MAPK inhibitors and led to the conclusion that PLC and to a lesser degree PI-3K activation are upstream events occurring in VPAC1 mediated PACAP stimulation of monocytes and are in contrast to ERK and p38 mandatory for the initiation of other cellular events associated with monocytes activation.