Multipotential neural stem cells (NSCs) in the central nervous system (CNS) proliferate indefinitely and give rise to neurons, astrocytes, and oligodendrocytes. As NSCs hold promise for CNS regeneration, it is important to understand how their proliferation and differentiation are controlled. We show here that the expression of sox2 gene, which is essential for the maintenance of NSCs, is regulated by the Gli2 transcription factor, a downstream mediator of sonic hedgehog (Shh) signaling: Gli2 binds to an enhancer that is vital for sox2 expression in telencephalic neuroepithelial (NE) cells, which consist of NSCs and neural precursor cells. Overexpression of a truncated form of Gli2 (Gli2⌬C) or Gli2-specific short hairpin RNA (Gli2 shRNA) in NE cells in vivo and in vitro inhibits cell proliferation and the expression of Sox2 and other NSC markers, including Hes1, Hes5, Notch1, CD133, and Bmi1. It also induces premature neuronal differentiation in the developing NE cells. In addition, we show evidence that Sox2 expression decreases significantly in the developing neuroepithelium of Gli2-deficient mice. Finally, we demonstrate that coexpression of Gli2⌬C and Sox2 can rescue the expression of Hes5 and prevent premature neuronal differentiation in NE cells but cannot rescue its proliferation. Thus these data reveal a novel transcriptional cascade, involving Gli2 3 Sox2 3 Hes5, which maintains the undifferentiated state of telencephalic NE cells.
Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N 6 ,2 -O-dibutyryl cAMP (Bt 2 AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt 2 AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt 2 AMP/theophylline. Taken together, these results indicated that Bt 2 AMP/ theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.Astrocyte differentiation occurs largely during the postnatal period (1), and the timing of the differentiation is regulated by various extracellular cues and cell-intrinsic programs (2). The extracellular stimuli known to promote astrocyte differentiation are fetal calf serum (3), bone morphogenic protein 2 (4), a neuropeptide, pituitary adenylate cyclase-activating polypeptide (5), and the interleukin-6 (IL-6) 1 family of cytokines (6). The IL-6 family of cytokines, oncostatin M and cardiotrophin-1, are known to activate a downstream transcription factor, signal transducer and activator of transcription 3 (STAT3), and then the activated STAT3 binds to the promoter region of the glial fibrillary acidic protein (GFAP) gene, an astrocyte marker, thereby inducing the expression of GFAP (7,8). STAT3 belongs to the STAT family of transcription factors, which play crucial roles in various intracellular signaling cascades involved in proliferation and differentiation (9, 10). In response to activation of cell-surface receptors, STAT3 is phosphorylated on its Tyr 705 residue by Janus kinase (JAK), and this leads to STAT3 dimerization, subsequent nuclear translocation, and the transactivation of its target genes (11). Bone morphogenic protein 2 synergistically acts with leukemia inhibitory factor, another IL-6 family of cytokines, to induce astrocytogenesis by promoting the complex formation of respective downstream transcription factors, Smads and STAT3, which are bridged by transcriptional coactivator p300 (12).The above-mentioned reports have extensively elucidated the mechanisms of cytokine...
Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme, contain cancer‐initiating cells (also known as cancer stem cells), which self‐renew and are malignant, with features of tissue‐specific stem cells. As these cells are resistant to irradiation and anti‐cancer drugs, it is important to characterize them and find targeting therapies. In this study, we established two primary human glioma cell lines from anaplastic oligodendroglioma and glioblastoma multiforme. These lines were enriched in glioma‐initiating cells, as just 10 cells formed malignant glioma when injected into mouse brain. We used these cell lines to examine the roles of the Notch, Hedgehog and Wnt signaling pathways, which are involved in stem‐cell maintenance and tumorigenesis, to determine which of these pathways are crucial to glioma‐initiating cells and their regulation. Here we show that the Hedgehog pathway is indispensable for glioma‐initiating cell proliferation and tumorigenesis; the Hedgehog signaling inhibitors prevented glioma‐initiating cell proliferation, while signaling inhibitors for Notch or Wnt did not. Overexpression of Gli2ΔC, a C‐terminal‐truncated form of Gli2 that antagonizes Gli transcription factor functions, blocked glioma‐initiating cell proliferation in culture and tumorigenesis in vivo. Knockdown of the Gli downstream factor Cdc2 also prevented glioma‐initiating cell proliferation. Taken together, these results show that the Hedgehog→ Gli→ Cdc2 signaling cascade plays a role in the proliferation and malignancy of glioma‐initiating cells. (Cancer Sci 2011; 102: 1306–1312)
PKN is a fatty acid-and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein ␣-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and ␣-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoAbinding domain was sufficiently shown to associate with ␣-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type ␣-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type ␣-actinin in a Ca 2؉ -sensitive manner and bound to that of skeletal muscle type ␣-actinin in a Ca 2؉ -insensitive manner. ␣-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and ␣-actinin lacking the actin-binding domain. In vitro translated full-length ␣-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4,5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length ␣-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and ␣-actinin.PKN is a serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family in the C terminus and a unique regulatory region in the N terminus (1, 2). The N-terminal region of PKN contains repeats of leucine zipper-like motif, suggesting promotion of protein-protein association through hydrophobic interactions (3). We demonstrated that Rho, a small GTP-binding protein, binds to PKN in a GTP-dependent fashion and that this binding leads to the activation of PKN, suggesting that PKN is one of the targets of Rho (4, 5). In order to identify other proteins that interact with PKN, we have used a yeast two-hybrid system with the Nterminal region of PKN as bait. One of the positive cDNA clones isolated from human brain cDNA library encoded a neurofilament L protein, a neuron-specific intermediate filament protein (6). We have demonstrated that PKN binds to and phosphorylates the head-rod domain of intermediate filament proteins such as each subunit of neurofilament and vimentin in vitro (6) and raised the possibility that PKN plays a role in the assembly of intermediate filament, one of the major components of cytoskeleton. Here we report that the two other groups of positive cDNA clones encoded ␣-actinin, a constituent of the other major component of cytoskeleton. MATERIALS AND METHODS Two-hybrid Screens and Constructs for Two-hybrid Systems-Schemes of the fusion constructs for human PKN...
Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42°C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37°C following heat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN. PKN is a fatty acid-activated serine/threonine protein kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and contains a unique regulatory region in the amino terminus (1-3). Recently, we demonstrated that Rho, a small GTPase protein, binds to PKN in a GTP-dependent fashion, and that this binding leads to the activation of PKN (4,5), suggesting that PKN is one of the targets of Rho. Rho is implicated in the organization of cytoskeleton in response to growth factors such as the formation of stress fibers and focal adhesions. Rho regulates cytoskeletal rearrangements, such as cell morphology (6), platelet aggregation (7,8), cell motility (9), and cytokinesis (10, 11). Rho also has roles in signaling to the nucleus and the regulation of transcriptional activation (12), cell-cycle progression (13), and cell transformation (14). Thus, the targets of the signaling pathway of Rho seem to be located within several cellular compartments. Signal transduction therefore requires the localization of Rho and Rho-regulated signaling molecules in each subcellular compartment that contains physiologically relevant roles mediated by Rho. We have reported that PKN associates and phosphorylates the intermediate filament proteins in vitro, indicating that the regulation of the cytoskeletal components was one of the possible functions of PKN (15).Recently, increasing evidence indicates that there is overlapping of the growth factor-and stress-signaling pathways. Rac and Cdc42Hs, other members of the Rho family small GTPases, are activated not only by growth factors but by stresses such as proinflammatory cytokines and ultraviolet radiation, and contribute to activation of stress-activated mitogen-activating protein kinases (16)(17)(18). However, little is known about the Rho-mediated signaling pathways of stresses. In this report, we investigate the effects of various stresses on the subcellular localization of PKN in culture cells and present a possibility that PKN, one of the targets of Rh...
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