Correlation between structure of Bcl-2 and its inhibitory function of JNK and caspase activity in dopaminergic neuronal apoptosis

To examine the correlation between the structure of Bcl-2 and its inhibitory function of c-Jun N-terminal kinase (JNK) and caspase activity, we established a dopaminergic neuronal cell line, MN9D overexpressing Bcl-2 (MN9D/Bcl-2) or its structural mutants. The mutants comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A; MN9D/BH2) domain and a deletion mutation in the C-terminal (MN9D/C22), BH3 (MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal deoxynucleotidyltransferase nick end-labeling) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, apoptotic death of MN9D/Neo cells reached 80-90% within 24 h in response to 1 microM staurosporine. Upon staurosporine treatment, JNK activity increased six- to sevenfold over the basal level within 2-4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z-VAD, attenuated cell death without suppressing JNK activation. Both staurosporine-induced cell death and JNK activation were attenuated in MN9D/Bcl-2. As determined by cleavage of poly(ADP-ribose) polymerase into 85 kDa, Bcl-2 blocked caspase activity as well. When cells overexpressing one of the Bcl-2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl-2 to JNK and caspase activity in staurosporine-induced dopaminergic neuronal cell death.     

Involvement of corticotropin-releasing factor receptor 2 beta in differentiation of dopaminergic MN9D cells

Corticotropin releasing factor (CRF) mediates various responses to stress through CRF receptors 1 and 2. CRF receptor 2 has two forms, 2alpha and 2beta each of which appears to have distinct roles. Here we used dopaminergic neuron-derived MN9D cells to investigate the function of CRF receptor 2 in dopamine neurons. We found that n-butyrate, a histone deacetylase inhibitor, induced MN9D cell differentiation and increased gene expression of all CRF receptors. CRF receptor 2beta was minimally expressed in MN9D cells; however, its expression dramatically increased during differentiation. CRF receptor 2beta expression levels appeared to correlate with neurite outgrowth, suggesting CRF receptor 2beta involvement in neuronal differentiation. To validate this statement, we made a CRF receptor 2beta-overexpressing MN9D/CRFR2 beta stable cell line. This cell line showed robust neurite outgrowth and GAP43 overexpression, together with MEK and ERK activation, suggesting MN9D cell neuronal differentiation. From these results, we conclude that CRF receptor 2beta plays an important role in MN9D cell differentiation by activating the MEK/ERK signaling pathway.     

Sequential Cleavage of Poly(ADP-Ribose)polymerase and Appearance of a Small Bax-Immunoreactive Protein Are Blocked by Bcl-XL and Caspase Inhibitors During Staurosporine-Induced Dopaminergic Neuronal Apoptosis

To assess the role of Bcl-X(L) and its splice derivative, Bcl-X(S), in staurosporine-induced cell death, we used a dopaminergic cell line, MN9D, transfected with bcl-xL (MN9D/Bcl-X(L)), bcl-xS (MN9D/Bcl-X(S)), or control vector (MN9D/Neo). Only 8.6% of MN9D/Neo cells survived after 24 h of 1 microM staurosporine treatment. Caspase activity was implicated because a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), attenuated staurosporine-induced cell death. Bcl-X(L) rescued MN9D cells from death (89.4% viable cells), whereas Bcl-X(S) had little or no effect. Bcl-X(L) prevented morphologically apoptotic changes as well as cleavage of poly(ADP-ribose)polymerase (PARP) induced by staurosporine. It is interesting that a small Bax-immunoreactive protein appeared 4-8 h after PARP cleavage in MN9D/Neo cells. The appearance of the small Bax-immunoreactive protein, however, may be cell type-specific as it was not observed in PC12 cells after staurosporine treatment. The sequential cleavage of PARP and the appearance of the small Bax-immunoreactive protein in MN9D cells were blocked either by Z-VAD-fmk or by Bcl-X(L). Thus, our present study suggests that Bcl-X(L) but not Bcl-X(S) prevents staurosporine-induced apoptosis by inhibiting the caspase activation that may be directly or indirectly responsible for the appearance of the small Bax-immunoreactive protein in some types of neurons.     

Regulatory Role of the JNK-STAT1/3 Signaling in Neuronal Differentiation of Cultured Mouse Embryonic Stem Cells

Stem cell transplantation therapy has provided promising hope for the treatment of a variety of neurodegenerative disorders. Among challenges in developing disease-specific stem cell therapies, identification of key regulatory signals for neuronal differentiation is an essential and critical issue that remains to be resolved. Several lines of evidence suggest that JNK, also known as SAPK, is involved in neuronal differentiation and neural plasticity. It may also play a role in neurite outgrowth during neuronal development. In cultured mouse embryonic stem (ES) cells, we test the hypothesis that the JNK pathway is required for neuronal differentiation. After neural induction, the cells were plated and underwent differentiation for up to 5 days. Western blot analysis showed a dramatic increase in phosphorylated JNKs at 1–5 days after plating. The phosphorylation of JNK subsequently induced activation of STAT1 and STAT3 that lead to expressions of GAP-43, neurofilament, βIII-tubulin, and synaptophysin. NeuN-colabelled with DCX, a marker for neuroblast, was enhanced by JNK signaling. Neuronal differentiation of ES cells was attenuated by treatment with SP600125, which inhibited the JNK activation and decreased the activation of STAT1 and STAT3, and consequently suppressed the expressions of GAP-43, neurofilament, βIII-tubulin, and the secretion of VEGF. Data from immunocytochemistry indicated that the nuclear translocation of STAT3 was reduced, and neurites of ES-derived neurons were shorter after treatment with SP600125 compared with control cells. These results suggest that the JNK-STAT3 pathway is a key regulator required for early neuronal differentiation of mouse ES cells. Further investigation on expression of JNK isoforms showed that JNK-3 was significantly upregulated during the differentiation stage, while JNK-1 and JNK-2 levels decreased. Our study provided interesting information on JNK functions during ES cell neuronal differentiation.     

SP600125对大鼠肺缺血/再灌注损伤的保护作用及机制

目的:探讨SP600125-c-Jun氨基末端激酶(JNK)特异性抑制剂对大鼠肺缺血/再灌注损伤的保护作用及机制。方法:复制在体大鼠原位单肺缺血/再灌注模型,随机分3组(n=10):假手术对照组(Control组)、缺血再灌注组(I/R组)与缺血再灌注+SP600125干预组(SP600125组)。实验结束时取肺组织测湿/干重比(W/D)、肺泡损伤率(IAR);采用蛋白印迹法检测肺组织磷酸化JNK(p-JNK)、JNK蛋白的表达;免疫组化法检测肺组织Bcl-2、Bax、Caspase-3蛋白的表达;原位末端标记法检测肺组织细胞凋亡指数(AI);电镜观察肺组织超微结构的改变。结果:SP600125组肺组织p-JNK、Bax、caspase-3的蛋白表达显著低于I/R组(均P<0.01),Bcl-2的蛋白表达及Bcl-2/Bax的比值显著高于I/R组(均P<0.01),AI、W/D及IAR显著低于I/R组(均P<0.01),肺组织超微结构损伤不同程度减轻。结论:SP600125可能通过抑制JNK信号通路,上调Bcl-2/Bax的比值减少caspase-3依赖性的肺细胞凋亡,从而减轻肺缺血/再灌注损伤。     

Cardiotoxin III induces c-jun N-terminal kinase-dependent apoptosis in HL-60 human leukaemia cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. The molecular effects of CTX III on HL-60 cells were dissected in the present study. We found that the antiproliferative action of CTX III on HL-60 cells was mediated through apoptosis, as characterized by an increase of sub G1 population, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Upregulation of Bax, downregulation of Bcl-2, the release of mitochondrial cytochrome c to cytosol and the activations of capase-9 and -3 were noted, while CTX III had no appreciable effect on the levels of Bcl-X(L) and Bad proteins. Moreover, c-Jun N-terminal kinase (JNK) was activated shortly after CTX III treatment in HL-60 cells. Consistently, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, suppressed apoptosis induced by CTX III. As expected, this JNK inhibitor also attenuated the modulation of Bax and Bcl-2, as well as the cytosolic appearance of cytochrome c and the activation of caspase-3 and caspase-9 that induced by CTX III. These findings suggest that CTX III can induce apoptosis in HL-60 cells via the mitochondrial caspase cascade and the activation of JNK is critical for the initiation of the apoptotic death of HL-60 cells.     

Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

核受体相关因子1表达下调对多巴胺能细胞酪氨酸羟化酶和神经突起生长的影响

将合成的核受体相关因子1（nuclear receptor-related factor 1,Nurr1）特异性短发夹寡核苷酸（small-hairpin RNA,shRNA）序列插入真核表达载体pSilen Circle（pSC）,构建Nurr1基因特异性shRNA真核表达载体,转染体外培养多巴胺能神经前体细胞系MN9D,分别采用实时荧光定量PCR和Western blot方法检测其对MN9D细胞内源Nurr1的干扰作用及其对酪氨酸羟化酶（tyrosine hydroxylase,TH）表达的影响,并在倒置显微镜下观察MN9D细胞神经突起生长的情况,探讨Nurr1 shRNA表达载体对多巴胺能细胞表型标记物删和以神经突起延长为特征的细胞成熟的影响。结果表明,脂质体组细胞和转染阴性对照质粒的MN9D细胞内Nurr1、TH的表达正常,而转染Nurr1 shRNA真核表达载体（pSC-N1和pSC-N2）的MN9D细胞内Nurr1和TH的mRNA水平明显降低,Nurr1 mRNA的下降率分别为62．3％和45．6％,TH mRNA的下降率分别为76.3％和62．6％。同时Nurr1和TH蛋白的表达亦明显下调,Nurr1蛋白的下降率分别为57．4％和72．0％,TH蛋白的下降率分别为79．1％和70．1％。另外,转染Nurr1 shRNA真核表达质粒的MN9D细胞神经突起延长有所减少,但是与正常细胞无明显差异。结果提示：Nurr1 shRNA真核表达载体能显著下调MN9D细胞内源Nurr1和TH mRNA和蛋白的表达,同时可能对MN9D细胞的神经突起延长有一定的抑制作用。Nurr1 shRNA表达载体的成功构建为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础。     

Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.     

JNK phosphorylation of paxillin, acting through the Rac1 and Cdc42 signaling cascade, mediates neurite extension in N1E-115 cells

Neurons extend neurites from the cell body before formation of the polarized processes of an axon and dendrites. Neurite outgrowth involves remodeling of the cytoskeletal components, which are initially regulated by small GTPases of the Rho family. Here we show that c-Jun N-terminal kinase (JNK), which is controlled by Rho GTPases Rac1 and Cdc42, is activated following neurite extension in mouse N1E-115 neuroblastoma cells as a model. The extension is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) and Clostridium difficile Toxin B, the inhibitor for Rho GTPases. Additionally, paxillin, the multifunctional focal adhesion protein, is phosphorylated at Ser 178 by upregulation of the Rac1/Cdc42/JNK cascade. Conversely, transfection of the paxillin construct harboring the Ser 178-to-Ala mutation into cells inhibits neurite extension. Taken together, these results suggest the novel role of the Rac1/Cdc42/JNK signaling cascade in neurite extension and indicate that the downstream target paxillin may be one of the convergent points of various signaling pathways underlying neurite extension.     

Distinct mechanisms of neurodegeneration induced by chronic complex I inhibition in dopaminergic and non-dopaminergic cells

Chronic mitochondrial dysfunction, in particular of complex I, has been strongly implicated in the dopaminergic neurodegeneration in Parkinson's disease. To elucidate the mechanisms of chronic complex I disruption-induced neurodegeneration, we induced differentiation of immortalized midbrain dopaminergic (MN9D) and non-dopaminergic (MN9X) neuronal cells, to maintain them in culture without significant cell proliferation and compared their survivals following chronic exposure to nanomolar rotenone, an irreversible complex I inhibitor. Rotenone killed more dopaminergic MN9D cells than non-dopaminergic MN9X cells. Oxidative stress played an important role in rotenone-induced neurodegeneration of MN9X cells, but not MN9D cells: rotenone oxidatively modified proteins more in MN9X cells than in MN9D cells and antioxidants decreased rotenone toxicity only in MN9X cells. MN9X cells were also more sensitive to exogenous oxidants than MN9D cells. In contrast, disruption of bioenergetics played a more important role in MN9D cells: rotenone decreased mitochondrial membrane protential and ATP levels in MN9D cells more than in MN9X cells. Supplementation of cellular energy with a ketone body, D-beta-hydroxybutyrate, decreased rotenone toxicity in MN9D cells, but not in MN9X cells. MN9D cells were also more susceptible to disruption of oxidative phosphorylation or glycolysis than MN9X cells. These findings indicate that, during chronic rotenone exposure, MN9D cells die primarily through mitochondrial energy disruption, whereas MN9X cells die primarily via oxidative stress. Thus, intrinsic properties of individual cell types play important roles in determining the predominant mechanism of complex I inhibition-induced neurodegeneration.     

BMP9经JNK激酶途径调控间充质干细胞C3H10T1/2成骨分化

为了证实JNK激酶在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9) 诱导间充质干细胞C3H10T1/2成骨分化中的作用,利用重组腺病毒将BMP9导入间充质干细胞C3H10T1/2. 通过碱性磷酸酶(ALP)活性测定、钙盐沉积实验、荧光素酶报告基因检测、Western印迹和组织化学染色等方法,检测BMP9是否可经JNK激酶途径调控间充质干细胞C3H10T1/2向成骨分化.动物实验验证在RNA沉默JNK蛋白激酶后,对BMP9诱导间充质干细胞C3H10T1/2向成骨分化的影响．结果发现,BMP9可以增强JNK 激酶的磷酸化;利用JNK抑制剂SP600125抑制JNK激酶活性后,BMP9诱导的间充质干细胞C3H10T1/2的早期成骨指标ALP活性和晚期指标钙盐沉积均受到抑制,而且经典SMAD信号的活化也相应受到抑制;RNA干扰沉默JNK基因表达后,同样也可抑制BMP9 诱导的C3H10T1/2细胞的ALP活性和裸鼠皮下异位成骨．因此表明,BMP9可活化JNK激酶途径从而诱导间充质干细胞C3H10T1/2向成骨分化．     

The prevention of the staurosporine-induced apoptosis by Bcl-XL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.     

Phospholipase D1 is an Important Regulator of bFGF-Induced Neurotrophin-3 Expression and Neurite Outgrowth in H19-7 Cells

The purpose of this study was to examine the role of phospholipase D1 (PLD1) in basic fibroblast growth factor (bFGF)-induced neurotrophin-3 (NT-3) expression and neurite outgrowth in H19-7 rat hippocampal neuronal progenitor cells. Overexpression of PLD1 increased bFGF-induced NT-3 expression, and dominant-negative-PLD1 or PLD1 siRNA abolished bFGF-induced NT-3 expression and neurite outgrowth. Treatment with bFGF activated the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway, and bFGF-induced NT-3 expression was blocked by a dominant-negative RhoA as well as by a specific Rho-kinase inhibitor (Y27632) and a SAPK/JNK inhibitor (SP600125). Furthermore, bFGF-induced JNK activation was also blocked by Y27632. These results indicate that the RhoA/ROCK/JNK pathway acts as an upstream signaling pathway in bFGF-induced NT-3 expression. Also, phosphatidic acid, the product of PLD, increased NT-3 expression. We found that PLD regulated the RhoA/ROCK/JNK pathway, which then led to Elk-1 transactivation. When Elk-1 activity was blocked by Elk-1 siRNA, bFGF-induced NT-3 expression and neurite outgrowth decreased. NT-3 overexpression increased neurite outgrowth, indicating that NT-3 is important for neurite outgrowth. Taken together, these results suggest that PLD1 is an important regulator of bFGF-induced NT-3 expression and neurite outgrowth, which are mediated by the RhoA/ROCK/JNK pathway via Elk-1 in H19-7 cells.     

Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line

The cell proliferation of p53-deficient Jurkat T cells is controlled after prolonged exposure to human lactoferrin (Lf). However, the molecular mechanism by which Lf influences these cellular responses remains unclear. In this study, we demonstrate that Lf-induced apoptosis in Jurkat T cells occurs in a dose- and time-dependent manner via the regulation of c-Jun N-terminal kinase (JNK) activity. Jurkat cells exposed to Lf for 1 day, especially at concentrations in excess of 500 microg/ml, showed typical apoptosis, as indicated by decreased cell viability and increased Annexin V binding. Our results also showed that Lf induced the activation of caspase 9 and caspase 3 activation, as demonstrated by our detection of cleaved caspases and PARP. Lf-induced apoptosis did not influence Bcl-2 expression via an ERK1/2 phosphorylation pathway, but was rather associated with the level of Bcl-2 phosphorylation. The treatment of cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, revealed that the JNK-Bcl-2 signaling cascade is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. These findings indicate that Lf functions as a biological mediator of apoptosis in the human leukemia Jurkat T-cell line, via the JNK-associated Bcl-2 signaling pathway.