Wnt信号通路关键基因β—catenin在RA诱导的P19胚胎性癌细胞神经分化 …

Ｗｎｔ信号参与了小鼠早期神经发育。我们以往的实验结果表明,Ｗｎｔ信号可引起Ｐ１９胚胎性癌细胞的神经分化。为进一步了解Ｗｎｔ信号在Ｐ１９神经分化过程中行使功能的时间,我们以Ｗｎｔ信号通路关键成员β－ｃａｔｅｎｉｎ是否定位在细胞核中作为考察Ｗｎｔ信号是否能传递到细胞核内调控下游基因活性的指标,分析了Ｗｎｔ信号在Ｐ     

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.     

Shiga toxins activate translational regulation pathways in intestinal epithelial cells

Shiga toxins (Stxs) cause irreversible damage to eukaryotic ribosomes, yet cellular intoxication of intestinal epithelial cells (IECs) results in increased synthesis of selected proteins, notably cytokines. How mRNA translation is maintained in this circumstance is unclear. This study was designed to assess whether Stx-induced alterations in host signal transduction machinery permit translation despite protein synthesis inhibition. A key step of translation is recruitment of initiation machinery to the 5' mRNA cap. This event occurs in part via interaction of the 5' cap with the cap binding protein, eIF4E, whose activity is positively regulated by phosphorylation and negatively regulated by binding to the translational repressor 4E-BP1. Following Stx treatment of IECs, eIF4E phosphorylation was detected by Western blotting using phospho-specific antibodies. Treatment with the p38 inhibitor, SB202190, or either of the ERK1/2 inhibitors, PD98059 and U0126, partially blocked Stx1-induced eIF4E phosphorylation. The Mnk1 inhibitor, CGP57380, blocked both basal and Stx-induced eIF4E phosphorylation. Interestingly, pretreatment with CGP57380 did not alter basal protein synthesis, but diminished the ability of cells to maintain translation following Stx1 challenge. Stx1 also induced hyperphosphorylation of 4E-BP1 and phosphorylation of S6Kinase; both effects were blocked by rapamycin. These data are novel observations showing that Stxs regulate multiple signal transduction pathways controlling translation in host cells, and support a role for eIF4E phosphorylation in maintaining host cell translation despite ribosomal intoxication.     

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.     

Regulation of protein abundance in pluripotent cells undergoing commitment to the neural lineage

The P19 cell line is a widely studied model of neural differentiation. When pluripotent P19 cells are cultured as aggregates in the presence of retinoic acid for 4 days, the cells commit to the neural fate, but have not yet undergone overt differentiation. Two-dimensional polyacrylamide gel electrophoresis was used to analyze cellular protein expression during this induction. Approximately 500 abundant polypeptides were analyzed. Seventeen polypeptides were upregulated during induction; several of these were significantly regulated 48 h after the addition of retinoic acid. No downregulations were observed. Fifteen of the 17 polypeptides continued to be expressed throughout terminal differentiation. The upregulation of 14 of the 17 polypeptides requires both retinoic acid and aggregation, which alone do not induce neural differentiation. Furthermore, these regulated polypeptides are expressed in neural tissue, suggesting they are associated with neural function in vivo. Embryonic stem cells, a totipotent line, also neurally differentiate in response to retinoic acid and aggregation. Comparison of embryonic stem cells to P19 cells shows that the two systems regulate a similar set of polypeptides and are thus likely to utilize a similar pathway. These studies are a step toward determining the full extent of regulation involved in the commitment of pluripotent cells to the neural fate. © 1996 Wiley-Liss, Inc.     

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. signal transduction; atrophy; autophagy     

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1

Protein synthesis is essential for the stabilization of glutamate receptor-dependent forms of long-lasting hippocampal synaptic plasticity and for the consolidation of memory, but the signal transduction mechanisms that regulate translation factors during these processes are not well understood. As a first step towards understanding how translation is activated during synaptic plasticity, we investigated how the eukaryotic initiation factor 4E (eIF4E), a rate-limiting mRNA cap-binding protein, and its kinase, Mnk1, are regulated by protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and N-methyl-D-aspartate (NMDA) receptor activation in hippocampal area CA1. We found that treatment of mouse hippocampal slices with either phorbol ester, to activate PKC, or forskolin, to activate PKA, resulted in activation of Mnk1 and increased eIF4E phosphorylation that was dependent on extracellular signal-regulated kinase (ERK). Similarly, brief treatment of hippocampal slices with NMDA resulted in activation of Mnk1 and increased phosphorylation of eIF4E. The NMDA-induced activation of Mnk1 and increased phosphorylation of eIF4E were dependent on PKA and ERK, but not PKC, and were present in synaptoneurosome preparations. Immunohistochemical analysis revealed that the PKA- and ERK-dependent increases in Mnk1 activation induced by NMDA also occurred in dendrites. These findings identify a specific regulatory pathway that can couple NMDA receptor activation to translation initiation factors in the hippocampus, and may represent a mechanism for triggering dendritic protein synthesis during long-term potentiation and long-term memory formation.     

Protection against anoikis and down-regulation of cadherin expression by a regulatable beta-catenin protein

beta-Catenin signaling plays a key role in a variety of cellular contexts during embryonic development and tissue differentiation. Aberrant beta-catenin signaling has also been implicated in promoting human colorectal carcinomas as well as a variety of other cancers. To study the molecular and cellular biological functions of beta-catenin in a controlled fashion, we created a regulatable form of activated beta-catenin by fusion to a modified estrogen receptor (ER) ligand binding domain (G525R). Transfection of tissue culture cells with expression vectors encoding this hybrid protein allows the signal transduction function of beta-catenin to be induced by the synthetic estrogen, 4-hydroxytamoxifen, leading to regulated activation of a beta-catenin-lymphocyte enhancer-binding factor-dependent reporter gene as well as induction of endogenous cyclin D1 expression. The activation of ER-beta-catenin signaling rescues RK3E cells from anoikis and correlates with an increased phosphorylation of mitogen-activated protein kinase. The inhibition of anoikis by ER-beta-catenin can be abolished by a mitogen-activated protein kinase pathway inhibitor, PD98059. Evidence is also provided to show that ER-beta-catenin down-regulates cadherin protein levels. These findings support a key role for activated beta-catenin signaling in processes that contribute to tumor formation and progression.     

Estrogen and progesterone regulate the IL-6 signal transduction pathway in antibody secreting cells

Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and traslocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.     

Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF-2α) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of elF-2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of elF-2 that delivers charged initiator tRNA_i^Met to the ribosome. When the levels of elF-2·GTP·Met-tRNA_i^Met ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF-2 by the protein kinase GCN2 decreases the concentration of elF-2·GTP·Met-tRNA_i^Met complexes by inhibiting the guanine nucleotide exchange factor for elF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF-2.     

Interleukin-6-induced tyrosine phosphorylation of phospholipase C-gamma1 in PC12 cells

Phospholipase C (PLC)-gamma1 plays a pivotal role in the signal transduction pathway mediated by growth factors. In this study, we found that neurite outgrowth of pheochromocytoma (PC12) cells was significantly induced by interleukin-6 (IL-6). Stimulation of PC12 cells with IL-6 led to tyrosine phosphorylation of PLC-gamma1 in a dose- and time-dependent manner. IL-6 stimulation also increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Accumulation of total inositol phosphate as well as tyrosine phosphorylation of PLC-gamma1 was inhibited by the pretreatment of protein kinase inhibitors such as genistein and staurosporine. These results suggest that PLC-gamma1 may be involved in the signal transduction pathway of IL-6-induced PC12 cell differentiation.     

Morphogenesis of human endothelial cells is inhibited by DAB2 via Src

Disabled-2 (DAB2) is an adaptor protein implicated in signal transduction pathways and in protein traffic regulation. Here, we show that DAB2 is highly expressed in human endothelial cells. DAB2 silencing in endothelial cells by lentiviral-mediated small hairpin RNA expression affects cell migration and differentiation into capillary-like structures while increasing cell proliferation and viability. DAB2 knockdown causes activation of the Src-FAK signal pathway, extracellular-signal regulated kinase and c-Jun NH2-terminal kinase activation, and inhibition of p38 phosphorylation. In DAB2 silenced endothelial cells, pharmacological inhibition of Src with its specific inhibitor PP2 abolishes focal adhesion kinase activation and restores differentiation of endothelial cells. These results suggest that DAB2, via Src and focal adhesion signaling, plays a role in human endothelial cell function.