A systemic administration of NMDA induces immediate early gene pip92 in the hippocampus

Abstract : In the mammalian CNS, aspartate and glutamate are major excitatory amino acids, and their receptors are believed to mediate a wide range of physiological and pathological processes, including neurotransmission, plasticity, excitotoxicity, and various forms of neurodegeneration. The immediate early gene pip92 has been identified in serum-stimulated BALB/c 3T3 fibroblasts, activated T lymphocytes treated with cycloheximide, and fibroblast growth factor-stimulated hippocampal cells during neuronal differentiation. In this study we have demonstrated that pip92 is expressed in the mouse brain after a single intraperitoneal injection of NMDA. The distribution of pip92 mRNA levels in the NMDA-treated mouse brain was investigated using in situ RT-PCR. The region-specific activation of pip92 in the CNS was observed 3 h after NMDA injection, and high levels of pip92 mRNA were detected in the hippocampal dentate gyrus and piriform cortex regions. In addition, the activation of pip92 by NMDA was mediated by activation of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK) and p38 kinase, but not extracellular signal-regulated kinase (ERK) in the mouse hippocampus and immortalized rat hippocampal progenitor cells. This study suggests that pip92 is likely to play an important role in neuronal cell death induced by excitotoxic NMDA injury in the CNS.     

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Abstract

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H₂O₂ accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.     

Expression of immediate early gene pip92 during anisomycin-induced cell death is mediated by the JNK- and p38-dependent activation of Elk1.

We report here that immediate early gene pip92 is expressed during anisomycin-induced cell death in fibroblast NIH3T3 cells. To determine the mechanism by which this occurs and to identify downstream signaling pathways, we investigated the induction of the pip92 promoter. The activation of pip92 by anisomycin is mediated by the activation of MAP kinases, such as JNK and p38 kinase, but not ERK. Deletion analysis of the pip92 promoter indicated that pip92 activation occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required for anisomycin-induced pip92 expression. Elk1, which binds to the Ets site, was phosphorylated by the JNK- and p38-dependent pathways and the phosphorylation of Elk1-GAL4 fusion proteins by these pathways was sufficient for the transactivation. Overall, this study suggested that different MAPK pathways are involved in the expression of immediate early gene pip92 by growth factors and environmental stresses.     

Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve

To clarify the presence of the Rho family of small GTPases p21-activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co-immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co-immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42-pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.     

Hyaluronic acid induces transglutaminase II to enhance cell motility; role of Rac1 and FAK in the induction of transglutaminase II

The role of transglutaminase II (TGase II) in hyaluronic acid (HA)-promoted melanoma cell motility was investigated. HA induced the expression of TGase II via the nuclear factor κB (NF-kB) in melanoma cells. HA increased the Rac1 activity and phosphorylation of focal adhesion kinase (FAK). Transfection by lipofectamine of dominant-negative Rac1, and FAK-related non-kinase (FRNK), an endogenous inhibitor of FAK, suppressed the induction of TGase II. This suggests that Rac1 and FAK mediate induction of TGase II by HA. HA-promoted melanoma cell motility was inhibited by cystamine, an inhibitor of TGase II, and overexpression of TGase II enhanced melanoma cell motility through reactive oxygen species. Taken together, HA promotes melanoma cell motility through activation of Rac1, FAK, and induction of TGase II.     

Involvement of Rac GTPase activation in phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to ICAM-1

Increasing evidence indicates that phospholipid oxidation plays important roles in atherosclerosis. Here, we investigated the involvement of Rho-family GTPases inphosphatidylcholine hydroperoxide (PCOOH)-induced THP-1 cell adhesion to ICAM-1. Isoprenoid depletion by fluvastatin and geranylgeranyltransferase inhibition by GGTI-286 suppressed PCOOH-induced cell adhesion to ICAM-1 and F-actin-rich membrane protrusion formation. Pull-down assays demonstrated the activation of Rac1 and Rac2 in PCOOH-treated cells. Pan-Rho-family GTPase inhibitor Clostridium difficile toxin B, Rac-specific inhibitor NSC23776, and RNA interference of the Rac isoforms suppressed the cell adhesion. These findings indicate the involvement of Rac GTPase activation in PCOOH-induced cell adhesion to ICAM-1 via actin reorganization.     

Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement

Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.     

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

The intracellular domain of L1CAM binds to casein kinase 2α and is neuroprotective via inhibition of the tumor suppressors PTEN and p53

Cell adhesion molecule L1 promotes neuritogenesis and neuronal survival through triggering MAPK pathways. Based on the findings that L1 is associated with casein kinase 2 (CK2), and that deficiency in PTEN promotes neuritogenesis in vitro and regeneration after trauma, we examined the functional relationship between L1 and PTEN. In parallel, we investigated the tumor suppressor p53, which also regulates neuritogenesis. Here, we report that the intracellular domain of L1 binds to the subunit CK2α, and that knockdown of L1 leads to CK2 dephosphorylation and an increase in PTEN and p53 levels. Overexpression of L1, but not the L1 mutants L1 (S1181N, E1184V), which reduced binding between L1 and CK2, reduced expression levels of PTEN and p53 proteins, and enhanced levels of phosphorylated CK2α and mammalian target of rapamycin, which is a downstream effector of PTEN and p53. Treatment of neurons with a CK2 inhibitor or transfection with CK2α siRNA increased levels of PTEN and p53, and inhibited neuritogenesis. The combined observations indicate that L1 downregulates expression of PTEN and p53 via direct binding to CK2α. We suggest that L1 stimulates neuritogenesis by activating CK2α leading to decreased levels of PTEN and p53 via a novel, L1‐triggered and CK2α‐mediated signal transduction pathway.

     

The N-BAR domain protein,Bin3, regulates Rac1- and Cdc42-dependent processes in myogenesis

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.     

A role for FXR and human FGF-19 in the repression of paraoxonase-1 gene expression by bile acids

Paraoxonase-1 (PON1), an enzyme that metabolizes organophosphate insecticides, is secreted by the liver and transported in the blood complexed to HDL. In humans and mice, low plasma levels of PON1 have also been linked to the development of atherosclerosis. We previously reported that hepatic Pon1 expression was decreased when C57BL/6J mice were fed a high-fat, high-cholesterol diet supplemented with cholic acid (CA). In the current study, we used wild-type and farnesoid X receptor (FXR) null mice to demonstrate that this repression is dependent upon CA and FXR. PON1 mRNA levels were also repressed when HepG2 cells, derived from a human hepatoma, were incubated with natural or highly specific synthetic FXR agonists. In contrast, fibroblast growth factor-19 (FGF-19) mRNA levels were greatly induced by these same FXR agonists. Furthermore, treatment of HepG2 cells with recombinant human FGF-19 significantly decreased PON1 mRNA levels. Finally, deletion studies revealed that the proximal -230 to -96 bp region of the PON1 promoter contains regulatory element(s) necessary for promoter activity and bile acid repression. These data demonstrate that human PON1 expression is repressed by bile acids through the actions of FXR and FGF-19.     

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.