Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.     

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.     

Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade

Adiponectin is the most abundant adipokine secreted from adipocytes. Accumulating evidence suggests that the physiological roles of adiponectin go beyond its metabolic effects. In the present study, we demonstrate that adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) are expressed in adult hippocampal neural stem/progenitor cells (hNSCs). Adiponectin treatment increases proliferation of cultured adult hNSCs in a dose- and time-dependent manner, whereas apoptosis and differentiation of adult hNSCs into neuronal or glial lineage were not affected. Adiponectin activates AMP-activated protein kinase and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in adult hNSCs. Pretreatment with the p38MAPK inhibitor SB203580, but not the AMP-activated protein kinase inhibitor Compound C, attenuates adiponectin-induced cell proliferation. Moreover, adiponectin induces phosphorylation of Ser-389, a key inhibitory site of glycogen synthase kinase 3β (GSK-3β), and this effect can be blocked by inhibition of p38MAPK with SB203580. Levels of total and nuclear β-catenin, the primary substrate of GSK-3β, were increased by adiponectin treatment. These results indicate that adiponectin stimulates proliferation of adult hNSCs, via acting on GSK-3β to promote nuclear accumulation of β-catenin. Thus, our studies uncover a novel role for adiponectin signaling in regulating proliferation of adult neural stem cells.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

GSK-3β-induced ASK1 stabilization is crucial in LPS-induced endotoxin shock

Glycogen synthase kinase-3β (GSK-3β), a multifunctional kinase, is a regulator of lipopolysaccharide (LPS)-mediated septic shock. Apoptosis signal-regulating kinase 1 (ASK1) is also required for LPS-induced activation of p38, which is a crucial determinant for the production of pro-inflammatory cytokines via Toll-like receptor 4 (TLR4) in endotoxemia. Here, we show that attenuation of endotoxemia induced by GSK-3 inhibition is caused by the ASK1 reduction-mediated inhibition of p38, a representative downstream kinase of ASK1. LPS-stimulated activation of p38 was blocked by the reduction of ASK1 via the knockdown of GSK-3β. In addition, compared with L929 control cells, ASK1 protein was reduced in L929 cells stably expressing Wnt-3a and in which β-catenin was active, due to the inhibition of GSK-3β activity. GSK-3β inhibition-mediated ASK1 reduction was also confirmed by reduced ASK1 in GSK-3β-deficient mouse embryo fibroblasts (MEFs) and MCF7 GSK-3β siRNA cells. Furthermore, ASK1 protein stability was also attenuated in MCF7 GSK-3β siRNA cells compared with GFP control cells. Consistent with stability data, a much stronger ubiquitination of ASK1 was observed in cells in which GSK-3β was knocked down. These findings suggest that GSK-3β crosstalks with p38 kinase via the regulation of ASK1 protein stability in endotoxemia.     

BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.     

Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation

Cells differentiate in response to various extracellular stimuli. This cellular response requires intracellular signaling pathways. The mitogen-activated protein (MAP) kinase cascade is a core signal transduction pathway that determines the fate of many kinds of cell. MAP kinase kinase kinase activates MAP kinase kinase, which in turn activates MAP kinase. Apoptosis signal-regulating kinase (ASK1) was identified as a MAP kinase kinase kinase involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Expression of the constitutively active form of ASK1 (ASK1-DeltaN) in keratinocytes induced significant morphological changes and differentiation markers, transglutaminase-1, loricrin, and involucrin. A transient increase in p21(Cip1/WAF1) reduced DNA synthesis, and cell cycle analysis verified the differentiation. p38 MAP kinase inhibitors, SB202190 and SB203580, abolished the induction of differentiation markers, transglutaminase-1, loricrin, and involucrin. In turn, the induction of differentiation with ceramide in keratinocytes caused an increase in ASK1 expression and activity. Furthermore, normal human skin expresses ASK1 protein in the upper epidermis, implicating ASK1 in in vivo keratinocyte differentiation. We propose that the ASK1-p38 MAP kinase cascade is a new intracellular regulator of keratinocyte differentiation.     

A chemical genomics screen to discover genes that modulate neural stem cell differentiation

The authors designed a chemical genomics screen with the aim of understanding genes and pathways that modulate neural stem/precursor cell differentiation. Multipotent mouse neural precursor cells isolated from cortices of embryonic day 12 (E12) embryos were subjected to spontaneous differentiation triggered by growth factor withdrawal. A quantitative whole-well immunofluorescence assay was set up to screen tool compound sets to identify small molecules with potent, dose-dependent, and reproducible effects on increasing neural stem cell differentiation toward neuronal lineage. Among the pro-neuronal compounds, kinase inhibitors were shown to exert pro-neuronal effect via a signaling pathway associated with the kinase. The global effect of hit compounds on modulating neuronal differentiation was confirmed by an in vivo mouse study and human neural stem cells culture. This study demonstrates that a phenotypic assay using cell type-specific antibody markers can be used for a large-scale compound screen to discover targets and pathways with impacts on differentiation of lineage-restricted precursor cells toward specific lineages.     

AICAR induces astroglial differentiation of neural stem cells via activating the JAK/STAT3 pathway independently of AMP-activated protein kinase

Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental, pathological, and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field, few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK), a metabolic "fuel gauge" of the biological system. In the present study, we found an unrecognized astrogliogenic activity of AICAR on not only immortalized neural stem cell line C17.2 (C17.2-NSC), but also primary neural stem cells (NSCs) derived from post-natal (P0) rat hippocampus (P0-NSC) and embryonic day 14 (E14) rat embryonic cortex (E14-NSC). However, another AMPK activator, Metformin, did not alter either the C17.2-NSC or E14-NSC undifferentiated state although both Metformin and AICAR can activate the AMPK pathway in NSC. Furthermore, overexpression of dominant-negative mutants of AMPK in C17.2-NSC was unable to block the gliogenic effects of AICAR. We also found AICAR could activate the Janus kinase (JAK) STAT3 pathway in both C17.2-NSC and E14-NSC but Metformin fails. JAK inhibitor I abolished the gliogenic effects of AICAR. Taken together, these results suggest that the astroglial differentiation effect of AICAR on neural stem cells was acting independently of AMPK and that the JAK-STAT3 pathway is essential for the gliogenic effect of AICAR.     

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.