Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFRalpha

The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor α (PDGFRα) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFRα. In this study, we analyzed the mechanism by which FIP1L1-PDGFRα induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFRα inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFRα induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.     

FGF2 stimulates the proliferation of human mesenchymal stem cells through the transient activation of JNK signaling

FGF2 has been shown to enhance proliferation and maintain differentiation potential in hMSCs during in vitro propagation. In this study, we investigated the role of mitogen-activated protein kinase in the functions of FGF2 in hMSCs. We demonstrated that FGF2 induces the transient activation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated protein kinase or p38 protein kinase. SP600125 and a dominant negative JNK1 significantly reduced the FGF2-enhanced proliferation of hMSCs. Treatment with SP600125 also diminished the activity of FGF2 in the maintenance of adipogenic and osteogenic differentiation potential. These results suggest that JNK signaling is involved in the FGF2-induced stimulation of the proliferation and the maintenance of differentiation potential in hMSCs.     

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.     

Pathway-specific profiling identifies the NF-kappa B-dependent tumor necrosis factor alpha-regulated genes in epidermal keratinocytes

Identification of tumor necrosis factor alpha (TNF alpha) as the key agent in inflammatory disorders led to new therapies specifically targeting TNF alpha and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNF alpha, drugs targeting TNF alpha have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNF alpha therapy. TNF alpha regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNF alpha signals to the nucleus. Hypothesizing that specific TNF alpha-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-kappa B-specific inhibitor, to identify the NF-kappa B-dependent set of the TNF alpha-regulated genes in human epidermal keratinocytes. Expression of approximately 40% of all TNF alpha-regulated genes depends on NF-kappa B; 17% are regulated early (1-4 h post-treatment), and 23% are regulated late (24-48 h). Cytokines and apoptosis-related and cornification proteins belong to the "early" NF-kappa B-dependent group, and antigen presentation proteins belong to the "late" group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-kappa B-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-kappa B pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-kappaB binding sites in their promoter DNA and are, presumably, directly regulated by NF-kappa B, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-kappa B-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNF alpha-dependent signaling pathway may inhibit a specific TNF alpha-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.     

Bcl-2 enhances neurite extension via activation of c-Jun N-terminal kinase

Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.     

c-Jun N-terminal kinase activity supports multiple phases of 3D-mammary epithelial acinus formation

Primary murine mammary epithelial cells cultured on a laminin-rich-extracellular matrix (ECM) require c-Jun N-terminal kinase (JNK) activity for acinus formation. Inhibition of JNK (using SP600125) or small interfering RNA-mediated knockdown of JNK1 blocked acinus formation, impaired cell polarisation and lumen clearance and allowed sustained extracellular signal-regulated kinase (ERK) phosphorylation, cell proliferation, adhesion-independent cell survival and expression of epithelial-mesenchymal transition markers. ERK inhibition abolished the effects of JNK blockade. Interestingly, inhibition of JNK from the time of cell seeding blocked cell polarisation and lumen clearance; later inhibition (≥ 6 h) only affected lumen clearance. ERK inhibition effectively protected cell polarisation but less so, lumen clearance. SP600125-treatment similarly affected acinus formation by the 'normal' human mammary epithelial MCF10A cell line. Expression of dominant-negative JNK1 in MCF10A cells also undermined acinus formation, generating large 'multi-acinar spheres' whose formation is probably driven by excessive luminal cell proliferation and cell survival. As JNK activity must be suppressed from the time of cell seeding to block cell polarisation, we studied the behaviour of MCF10A cells immediately after seeding in laminin rich matrix: we detected engagement of cells with the matrix, early polarisation, movement of cells into clusters and 'epithelial-cell- like' behaviour of clustered cells. Inhibition of JNK activity or expression of dominant-negative JNK1 allowed cell engagement to the matrix, but blocked cell polarisation and all subsequent 'behaviours'. While integrin activation occurred, tyrosine-phosphorylation of paxillin, Fak and Src was significantly damped by JNK inhibition. These results emphasise the multi-phase dependency of the organisation of mammary cells in 3D on JNK activity and suggest a 'permissive' support of ECM-integrin 'outside-in' signalling and a 'damping' of growth-factor ERK signalling as its two key cell physiological effects.     

Differentiation-associated genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in KYSE450 cells

A group of potential differentiation-associated genes had been identified by microarray analysis as c-Jun/AP-1 target genes essential for epithelial differentiation program. Our previous study showed that c-Jun/AP-1 could bind and activate these gene promoters in vivo using chromatin immunoprecipitation. To further understand how the mitogen-activated protein kinase signaling pathways regulate AP-1 activity and expression of c-Jun target genes, our strategy was based on the use of 12-o-tetradecanoylophorbol-13-acetate (TPA) and pharmacological reagents to induce or block c-Jun expression. The mRNA and protein expression of these genes increased in response to TPA-induced c-Jun/AP-1 expression. Inhibitors of JNK (SP600125) and PKC (GF109203X) mainly blocked expression and phosphorylation of c-Jun, while inhibition of MEK-ERK activity with PD98059 (an inhibitor of MEK) had little effect. Expression of involucrin and keratin 4 in response to TPA was attenuated by pretreatments with GF109203X and SP600125, but not PD98059, suggesting involvement of PKC and JNK in this response. Taken together, these results suggested that differentiation-associated genes were regulated by TPA-induced c-Jun/AP-1 mainly via a PKC/JNK pathway in esophageal cancer cell line KYSE450.     

Involvement of JNK in the Embryonic Development and Organogenesis in Zebrafish

c-Jun N-terminal kinase (JNK) is one of the mitogen-activated protein kinases. Previous studies showed that the JNK is involved in signaling pathways initiating cell cycle, and eventually, causing apoptosis through persistent activation in mammals. In this article, it is further revealed that the jnk1 gene is closely related with the embryonic development and organogenesis in zebrafish. RT-PCR and Western blot analysis show that there were distinct expression patterns of JNK at the different developmental stages as well as in the various tissues in zebrafish. Knockdown of jnk1 by RNA interference (RNAi) resulted in high lethal, serious retardation and malformations of embryos in zebrafish. SP600125, a JNK-specific inhibitor, gives rise to high mortality in zebrafish, similar to that caused by the jnk1 RNA interference. SP600125 is also responsible for the severe abnormality of organs, especially the skeletal system, such as skull, mandible deficiency, and cyrtosis heterauxesis. The results also indicate that the inhibition of JNK by SP600125 suppresses the ovarian differentiation during the embryo development in zebrafish. Overall, our study demonstrates that the jnk1 gene is required for ovary differentiation and development in the zebrafish, and down-regulated JNK directly inhibits ovary differentiation during early ontogenetic stages.     

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．     

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.