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.     

The isoform-specific functions of the c-Jun N-terminal Kinases (JNKs): differences revealed by gene targeting

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family. In mammalian genomes, three genes encode the JNK family. To evaluate JNK function, mice have been created with deletions in one or more of three Jnk genes. Initial studies on jnk1(-/-) or jnk2(-/-) mice have shown roles for these JNKs in the immune system whereas studies on jnk3(-/-) mice have highlighted roles for JNK3 in the nervous system. Further studies have highlighted the contributions of JNK1 and/or JNK2 to a range of biological and pathological processes. These include bone remodelling and joint disease, inflammatory and autoimmune diseases, obesity, diabetes, cardiovascular disease, liver disease and tumorigenesis in addition to effects in neurons. These results emphasise the differences in the roles played by JNK isoforms in vivo and suggest that the design of JNK inhibitors for subsequent therapeutic uses may benefit from selective inhibition of individual JNK isoforms.     

Beta-catenin signaling is required for neural differentiation of embryonic stem cells

Culture of embryonic stem (ES) cells at high density inhibits both beta-catenin signaling and neural differentiation. ES cell density does not influence beta-catenin expression, but a greater proportion of beta-catenin is targeted for degradation in high-density cultures. Moreover, in high-density cultures, beta-catenin is preferentially localized to the membrane further reducing beta-catenin signaling. Increasing beta-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression of beta-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, beta-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of beta-catenin. By contrast, RA does not induce neurogenesis in high-density cultures in the absence of beta-catenin signaling. Truncation of the armadillo domain of beta-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of beta-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by beta-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the beta-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon beta-catenin signaling. These observations suggest that beta-catenin signaling promotes neural lineage commitment by ES cells, and that beta-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of beta-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.     

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.     

MKK7γ1 reverses nerve growth factor signals: proliferation and cell death instead of neuritogenesis and protection

c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54 kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21^WAF1/CIP1, which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes.     

Coordination of JNK1 and JNK2 is critical for GADD45alpha induction and its mediated cell apoptosis in arsenite responses

Arsenite is a well documented environmental pathogen, whereas it has also been applied as medication to treat various neoplasmas. The pathogenic and therapeutic effects of arsenite are associated with cellular apoptotic responses. However, the molecular mechanisms of arsenite-induced apoptosis are not very well understood. Our previous study has shown that arsenite exposure is able to activate JNKs, which subsequently mediate the apoptotic outcome. The present study further revealed that the coordination of JNK1 and JNK2 was critical for the arsenite-induced expression of GADD45alpha (growth arrest and DNA damage 45alpha), which in turn mediated the cellular apoptosis. The arsenite-induced apoptosis and GADD45alpha expression were significantly impaired in mouse embryonic fibroblasts deficient in either jnk1 (JNK1-/-) or jnk2 (JNK2-/-). Knockdown of GADD45alpha by its specific small interfering RNA also dramatically reduced the apoptotic responses, and overexpression of GADD45alpha in either JNK1-/- or JNK2-/- mouse embryonic fibroblasts partially resensitized the cell death. Furthermore, it was found that the regulation of GADD45alpha by JNK1 and JNK2 was achieved through mediating the activation of c-Jun, since in the JNK1-/- and JNK2-/- cells the c-Jun activation was impaired, and overexpression of the dominant negative mutant of c-Jun (TAM67) in wild type cells could also block GADD45alpha induction as well as cellular apoptosis. Our results demonstrate that the coordination of JNK1 and JNK2 is critical for c-Jun/GADD45alpha-mediated cellular apoptosis induced by arsenite.     

The double inhibition of endogenously produced BMP and Wnt factors synergistically triggers dorsal telencephalic differentiation of mouse ES cells

Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high‐throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 66–79, 2015     

Caspase-mediated cleavage of JNK during stress-induced apoptosis

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). The JNKs are encoded by three separate genes (jnk1, jnk2, and jnk3), which are spliced alternatively to create 10 JNK isoforms that are either p46 or p54 in size. In this study, we found that the p52 form of JNK emerged in human leukemia MOLT-4 or U937 cells following X-irradiation or heat treatment. The accumulation of p52 coincided with the reduction of p54 JNK. On the other hand, the amounts of p46 JNK did not change by X-irradiation. Induction of the p52 form of JNK also paralleled the appearance of the active form of caspase-3 and was suppressed by a caspase-specific inhibitor, Ac-DEVD-CHO, but not by Ac-YVAD-CHO. In vitro cleavage assays indicated that recombinant human JNK1beta2 and JNK2beta2 were cleaved by caspase-3, and that the mutation of aspartic acid at position 413 of JNK1beta2 or 410 of JNK2beta2 to alanine abolished the cleavage. Altogether, our results demonstrated that p54 JNKs, at least JNK1beta2 and JNK2beta2, were new selective targets of caspases in JNK splicing variants, and suggested that the p52 form could serve as a marker of apoptosis.     

Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2

Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.     

Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca²⁺ mobilizing nucleotide presented in various species. NAADP mobilizes Ca²⁺ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.     

Genetic inhibition or activation of JNK1/2 protects the myocardium from ischemia-reperfusion-induced cell death in vivo

The c-Jun NH2-terminal kinase (JNK) branch of the mitogen-activated protein kinase signaling cascade has been implicated in the regulation of apoptosis in a variety of mammalian cell types. In the heart, disagreement persists concerning the role that JNKs may play in regulating apoptosis, since both pro- and antiapoptotic regulatory functions have been reported in cultured cardiomyocytes. Here we report the first analysis of cardiomyocyte cell death due to JNK inhibition or activation in vivo using genetically modified mice. Three separate mouse models with selective JNK inhibition were assessed for ventricular damage and apoptosis levels following ischemia-reperfusion injury. jnk1-/-, jnk2-/-, and transgenic mice expressing dominant negative JNK1/2 within the heart were each shown to have less JNK activity in the heart and less injury and cellular apoptosis in vivo following ischemia-reperfusion injury. To potentially address the reciprocal gain-of-function phenotype associated with sustained JNK activation, transgenic mice were generated that express MKK7 in the heart. These transgenic mice displayed elevated cardiac c-Jun kinase activity but, ironically, were also significantly protected from ischemia-reperfusion. Mechanistically, JNK-inhibited mice showed increased phosphorylation of the proapoptotic factor Bad at position 112, whereas MKK7 transgenic mice showed decreased phosphorylation of this site. Collectively, these results underscore the complexity associated with JNK signaling in regulating apoptosis, such that sustained inhibition or activation both elicit cellular protection in vivo, although probably through different mechanisms.     

c-Jun N-terminal kinase 2 (JNK2) enhances cell migration through epidermal growth factor substrate 8 (EPS8)

Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor substrate 8 (EPS8) expression, a key regulator of EGF receptor (R) signaling and trafficking. Using jnk2(-/-) mice, we found that EPS8 expression is higher in polyoma middle T antigen (PyVMT)jnk2(-/-) mammary tumors and jnk2(-/-) mammary glands compared with the respective jnk2(+/+) controls. The inverse relationship between the jnk2 and eps8 expression was also associated with cancer progression in that patients with basal-type breast tumors expressing high jnk2 and low eps8 experienced poor disease-free survival. In mammary tumor cell lines, the absence of jnk2 greatly reduces cell migration that is rescued by EPS8 knockdown. Subsequent studies show that JNK2 enhances formation of the EPS8-Abi-1-Sos-1 complex to augment EGFR activation of Akt and ERK, whereas the absence of JNK2 promotes ESP8/RN-Tre association to inhibit endocytotic trafficking of the EGFR. Together, these studies unveil a critical role for JNK2 and EPS8 in receptor tyrosine kinase signaling and trafficking to convey distinctly different effects on cell migration.     

Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.