Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinoma A 549 cells

The effect of the pan c-Jun N-terminal kinase (JNK) inhibitor SP600125 on the proliferation of human lung carcinoma A549 cells has been evaluated. We have shown that SP600125 completely inhibited the proliferation of A549 cells, the cycle arrest being in G2/M phase. When cells were treated with SP600125 for >12h, a cell population with DNA content of 4n to 8n was detected. Moreover, the effect of SP600125 on the expression of cell cycle related proteins was an upregulation of p53 protein accompanied by an increase in its molecular mass. Prolonged SP600125 treatment downregulated p21, Bax and Mdm2 expression, but increased the level of the cellular p53-Mdm2 complex. Taken together, we show that SP600125 could induce G2/M cell cycle arrest and endoreduplication in a p21 independent manner, and that SP600125 could also post-translationally modify p53 to modify its function. Our data show that basic JNK activity plays an important role in the progression of the cell cycle at G2/M cell phase.     

A JNK inhibitor SP600125 induces defective cytokinesis and enlargement in P19 embryonal carcinoma cells

While analyzing the role of c‐Jun NH₂‐terminal kinase (JNK) in neurogenesis in P19 embryonal carcinoma cells, we noticed that treatment with SP600125, a JNK inhibitor, increased the cell size markedly. SP600125‐induced enlargement of P19 cells was time‐ and dose‐dependent. The increased cell size in response to SP600125 was also detected in B6mt‐1 embryonic stem cells. SP600125 treatment inhibited cell growth and increased DNA contents, indicating the inhibition of cell proliferation resulting from endoreduplication. Concurrently, the gene expression of p21, a regulator of G2/M arrest as well as G1 arrest, was increased in cells treated with SP600125. The increased cell size in response to SP600125 was detected even in P19 cells treated with colcemide, an inhibitor of cell cycle progression at the metaphase. The present study suggests that treatment with SP600125 progresses the cell cycle, skipping cytokinesis in P19 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

SP600125孵育影响鲫骨骼发育基因表达

前期研究表明ATP竞争性小分子抑制剂SP600125在体外可以高效诱导鱼类细胞多倍化。研究首次尝试用SP600125直接孵育鲫受精卵以获得多倍体鱼的可能性。结果显示SP600125处理受精卵虽然能够引起部分胚胎倍性发生改变, 但也显著影响胚胎发育并导致胚胎高死亡率, 同时伴随着畸形鱼苗的发生, 其中主要表现为脊椎弯曲和尾缺失等骨骼发育异常。SP600125处理导致骨骼畸形在SP600125孵育鲫鱼苗试验中也同样获得验证。荧光定量PCR检测结果表明, 在体(受精卵和鱼苗)或离体(培养的尾鳍细胞)状态下, SP600125孵育会引起smad6、stat1、 lef1、bmp2和twist1等骨骼发育相关基因mRNA水平显著上调。相关结果为进一步调整SP600125诱导的鱼类倍性操作策略和方法提供了重要理论参考。     

Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.     

Identification of side population cells in mouse primordial germ cells and prenatal testis

In mammals, the stem cells of spermatogenesis are derived from an embryonic cell population called primordial germ cells (PGCs). Spermatogonial stem cells displaying the “side population” (SP) phenotype have been identified in the immature and adult mouse testis, but noting is known about the expression of the SP phenotype during prenatal development of germ cells. The SP phenotype, defined as the ability of cells to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In the present study, we analyzed and characterized the Hoechst SP via cytofluorimetric analysis of disaggregated gonads at different time points during embryonic development in mice. To directly test the hypothesis that the SP phenotype is a feature of germ cell lineage, experiments were performed on transgenic animals expressing enhanced green fluorescent protein (EGFP) under the control of the Oct4 promoter, to identify early germ cells up to PGCs. We found that prenatal gonads contain a fraction of SP cells at each stage analyzed, and the percentage of cells in the SP fraction decreases as development proceeds. Surprisingly, more than 50% of the PGCs displayed the SP phenotype at 11.5 dpc (days post coitum). The percentage of germ cells with the SP phenotype decreased steadily with development, to less than 1% at 18.5 dpc. Cytofluorimetric analysis along with immunocytochemistry performed on sorted cells indicated that the SP fraction of prenatal gonads, as in the adult testis, was heterogeneous, being composed of both somatic and germ cells. Both cell types expressed the ABC transporters Abcg2, Abcb1a, Abcb1b and Abcc1. These findings provide evidence that the SP phenotype is a common feature of PGCs and identifies a subpopulation of fetal testis cells including prospermatogonia whose differentiation fate remains to be investigated.     

SP600125 inhibits cap-dependent translation independently of the c-Jun N-terminal kinase pathway

We investigated the effects of SP600125 (formerly called c-Jun N-terminal kinase (JNK) inhibitor II) on translation using cultured mouse cells. SP600125 (50 μM) treatment rapidly repressed overall protein synthesis, accompanied by a reduction in the mRNAs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase in the polysomal fraction. SP600125 decreased polysomes with a concomitant increase in free ribosomal subunits in the cytoplasm, suggesting that global translation was inhibited at the initiation step. A reporter analysis using exogenous mRNAs showed that SP600125 inhibited cap-dependent but not internal ribosome entry site-dependent translation. SP600125 significantly attenuated phosphorylation of components in the mTOR pathway, which is responsible for cap-dependent translation. In contrast to SP600125, short hairpin RNAs for JNK1 and JNK2 failed to affect overall protein synthesis. Collectively, SP600125 inhibits cap-dependent translation, independent of the JNK pathway.     

Mouse ES cells maintained in different pluripotency-promoting conditions differ in their neural differentiation propensity

Prior to differentiation, embryonic stem (ES) cells in culture are maintained in a so-called “undifferentiated” state, allowing derivation of multiple downstream cell lineages when induced in a directed manner, which in turn grants these cells their “pluripotent” state. The current work is based on a simple observation that the initial culture condition for maintaining mouse ES cells in an “undifferentiated” state does impact on the differentiation propensity of these cells, in this case to a neuronal fate. We point out the importance in judging the “pluripotency” of a given stem cell culture, as this clearly demonstrated that the “undifferentiated” state of these cells is not necessarily a “pluripotent” state, even for a widely used mouse ES cell line. We partly attribute this difference in the initial value of ES cells to the naïve-to-primed status of pluripotency, which in turn may affect early events of the differentiation in vitro.     

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.     

Reprogramming cell fates: reconciling rarity with robustness

The stunning possibility of “reprogramming” differentiated somatic cells to express a pluripotent stem cell phenotype (iPS, induced pluripotent stem cell) and the “ground state” character of pluripotency reveal fundamental features of cell fate regulation that lie beyond existing paradigms. The rarity of reprogramming events appears to contradict the robustness with which the unfathomably complex phenotype of stem cells can reliably be generated. This apparent paradox, however, is naturally explained by the rugged “epigenetic landscape” with valleys representing “preprogrammed” attractor states that emerge from the dynamical constraints of the gene regulatory network. This article provides a pedagogical primer to the fundamental principles of gene regulatory networks as integrated dynamic systems and reviews recent insights in gene expression noise and fate determination, thereby offering a formal framework that may help us to understand why cell fate reprogramming events are inherently rare and yet so robust.     

The JNK inhibitor SP600125 enhances dihydroartemisinin-induced apoptosis by accelerating Bax translocation into mitochondria in human lung adenocarcinoma cells

The C-Jun N-terminal Kinase (JNK) inhibitor SP600125 is widely used to inhibit the JNK-mediated Bax activation and cell apoptosis. However, this report demonstrates that SP600125 synergistically enhances the dihydroartemisinin (DHA)-induced human lung adenocarcinoma cell apoptosis by accelerating Bax translocation and subsequent intrinsic apoptotic pathway involving mitochondrial membrane depolarization, cytochrome c release, caspase-9 and caspase-3 activation. The dynamical analysis of GFP-Bax mobility inside single living cells using fluorescence recovery after photobleaching revealed that SP600125 aggravated the DHA-induced decrease of Bax mobility and Bax translocation. These results for the first time present a novel pro-apoptotic action of SP600125 in DHA-induced apoptosis.     

SP600125, an inhibitor of c-jun N-terminal kinase,activates CREB by a p38 MAPK-mediated pathway

SP600125, an anthrapyrazolone inhibitor of c-jun N-terminal kinase (JNK), has been used to characterize the role of JNK in apoptotic pathways. In this study, we have demonstrated an additional novel anti-apoptotic action of this inhibitor in MIN6 cells, a mouse beta cell line. SP600125 induced CREB-dependent promoter activation by 2.8-fold at 20 microM, the concentration at which it inhibited c-jun-dependent promoter activation by 51%. There was a significant (P<0.01) increase in CREB phosphorylation (serine 133) at 5 min, which persisted for a period of 2h. Examination of signaling pathways upstream of CREB showed a 2.5-fold increase in the active phospho form of p38 MAPK. This finding was further confirmed by an in vitro kinase assay using ATF-2 as substrate. SB203580, an inhibitor of p38 MAPK, partially blocked SP600125-mediated activation of CREB. These observations suggest that SP600125 could be used as a small molecular weight activator of CREB.     

Ablation of the spindle assembly checkpoint by a compound targeting Mps1

The spindle assembly checkpoint ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are properly attached to the mitotic spindle. Here, we show that the previously reported c-Jun amino-terminal kinase (JNK) inhibitor SP600125 effectively disrupts spindle checkpoint function in a JNK-independent fashion. SP600125 potently inhibits activity of the mitotic checkpoint kinase monopolar spindle 1 (Mps1) in vitro and triggers efficient progression through a mitotic arrest imposed by spindle poisons. Importantly, expression of an Mps1 mutant protein refractory to SP600125-mediated inhibition restores spindle checkpoint function in the presence of SP600125, showing that its mitotic phenotype is induced by Mps1 inhibition in vivo. Remarkably, primary human cells are largely resistant to the checkpoint-inactivating action of SP600125, suggesting the existence of Mps1-independent checkpoint pathways that are compromised in tumour cells.     

Minimal Hodgkin–Huxley type models for different classes of cortical and thalamic neurons

We review here the development of Hodgkin–Huxley (HH) type models of cerebral cortex and thalamic neurons for network simulations. The intrinsic electrophysiological properties of cortical neurons were analyzed from several preparations, and we selected the four most prominent electrophysiological classes of neurons. These four classes are “fast spiking” “regular spiking” “intrinsically bursting” and “low-threshold spike” cells. For each class, we fit “minimal” HH type models to experimental data. The models contain the minimal set of voltage-dependent currents to account for the data. To obtain models as generic as possible, we used data from different preparations in vivo and in vitro, such as rat somatosensory cortex and thalamus, guinea-pig visual and frontal cortex, ferret visual cortex, cat visual cortex and cat association cortex. For two cell classes, we used automatic fitting procedures applied to several cells, which revealed substantial cell-to-cell variability within each class. The selection of such cellular models constitutes a necessary step towards building network simulations of the thalamocortical system with realistic cellular dynamical properties.     

Enteral arginine modulates inhibition of AP-1/c-Jun by SP600125 in the postischemic gut

We previously demonstrated that enteral arginine increased c-Jun/activator protein-1 (AP-1) DNA-binding activity and iNOS expression in a rodent model of mesenteric ischemia/reperfusion (I/R). The objective of this study was to specifically investigate the role of AP-1 in arginine's deleterious effect on the postischemic gut. We hypothesized that AP-1 inhibition would mitigate the effects of arginine. Using a rodent model of mesenteric I/R we demonstrated that gut neutrophil infiltration, activity of c-Jun/AP-1, as well as iNOS expression were increased by I/R and further increased by arginine while lessened by inhibition of c-Jun using the pharmacologic c-Jun N-terminal kinase inhibitor, SP600125. Similar results were demonstrated using a cell culture model of oxidant stress in IEC-6 cells. Importantly, effects of SP600125 were comparable to those of c-Jun silencing. Lastly, the specific iNOS inhibitor, 1400W, had no effect on either AP-1 or c-Jun. In conclusion, SP600125 attenuated the activity of c-Jun/AP-1, iNOS expression, and neutrophil infiltration induced by arginine following mesenteric I/R. Our data suggest that AP-1 inhibition mitigates the injurious inflammatory effects of arginine in the postischemic gut. Further investigation into the pathologic role of enteral arginine in the postischemic gut is warranted.     

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.     

TRB3 interacts with ERK and JNK and contributes to the proliferation,apoptosis, and migration of lung adenocarcinoma cells

Tribbles homolog 3 (TRB3) has been accounted for regulation of a few cell processes through interaction with other significant proteins. The molecular mechanisms underlying TRB3 in tumorigenesis in lung adenocarcinoma have not been entirely elucidated. The present study is aimed at determining the function and fundamental mechanisms of TRB3 in lung adenocarcinoma progression. TRB3 was highly expressed in A549 and H1299 cells and lung adenocarcinoma tissues compared with human bronchial epithelial cells (HBEpC) and adjacent normal lung tissues. Hypoxia significantly upregulated the expression of TRB3 protein in A549 and H1299 cells in a time-dependent way. Gene expression profiling interactive analysis data analysis indicated that patients with lung adenocarcinoma with excessive expression of TRB3 mRNA had fundamentally shorter survival time. TRB3 knockdown in A549 cells can inhibit cell proliferation and migration, and promote cell apoptosis. TRB3 knockdown reduced the expression of p-ERK and p-JNK, but did not affect the expression of p-P38 MAPK. TRB3 overexpression enhances the malignant transformation abilities of HBEpC such as cell proliferation, migration and colony formation, which could be reversed by U0126 and SP600125. TRB3 overexpression promotes the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but was not affected by U0126 and SP600125. The results of coimmunoprecipitation experiments indicated that TRB3 binds directly to ERK and JNK. This study suggests that TRB3 has a potentially carcinogenic role in lung adenocarcinoma by binding to ERK and JNK and promoting the phosphorylation of ERK and JNK. TRB3 can be a possible therapeutic focus for lung adenocarcinoma.     

SP600125, a selective JNK inhibitor, protects ischemic renal injury via suppressing the extrinsic pathways of apoptosis

Accumulating evidence suggests that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in renal ischemia/reperfusion injury. However, the downstream mechanism that accounts for the proapoptotic actions of JNK during renal ischemia/reperfusion has not been elucidated. We report that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion via suppression of the extrinsic pathway. This corresponds to the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3 h after renal ischemia. Additionally, SP600125 attenuated the increased expression of FasL induced by ischemia/reperfusion at 3 h. The administration of SP600125 prior to ischemia was also protective. Thus, our findings imply that SP600125 can inhibit the activation of the JNK-c-Jun-FasL pathway and protect renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for renal ischemia/reperfusion injury.     

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.