Neurons, astrocytes, and oligodendrocytes—the three major cell types that comprise the central nervous system—are generated from common multipotent neural precursor cells (NPCs). Members of the interleukin‐6 family of cytokines, including leukemia inhibitory factor (LIF), induce astrocyte differentiation of NPCs by activating the transcription factor signal transducer and activator of transcription 3 (STAT3). We show here that retinoic acid (RA) facilitates LIF‐induced astrocyte differentiation of NPCs. RA and LIF synergistically activate the promoter of gfap, which encodes the astrocytic marker glial fibrillary acidic protein, and a putative RA response element in the promoter was found to be critical for this activation. Histone H3 acetylation around the STAT‐binding site in the gfap promoter was increased in NPCs treated with RA, allowing STAT3 to gain access to the promoter more efficiently. These results suggest that RA acts in concert with LIF to induce astrocyte differentiation of NPCs through an epigenetic mechanism that involves cross‐talk between distinct signaling pathways. Stem Cells 2009;27:2744–2752
Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocytogenesis, which normally commences at later stages of development. We have previously reported that a particular cytosine residue within a STAT3-binding site in the astrocyte-specific marker glial fibrillary acidic protein (GFAP) gene promoter becomes demethylated in neuroepithelial cells as gestation proceeds. This demethylation correlates tightly with the onset of astrocyte differentiation, suggesting that a change in DNA methylation at cell-type-specific gene promoters controls the switch from neurogenesis to astrocytogenesis in the developing brain. Here, we show that late-gestation neuroepithelial cells, which have already lost the methylation in the STAT3-binding site within the GFAP promoter, can still give rise to neurons and that these neurons do not respond to a STAT3-activating cytokine to express GFAP. Members of a transcriptional repressor family, the methylated-CpG binding proteins (MBDs), including MeCP2, are predominantly expressed in neurons, and ectopic MeCP2 expression inhibited astrocyte differentiation of neuroepithelial cells. Moreover, we found that exon 1 of the GFAP gene remains hypermethylated even in neuroepithelial cells at a late developmental stage and in neurons differentiated from such neuroepithelial cells. We further demonstrate that MeCP2 actually binds to the highly methylated exon 1 of the GFAP gene in neurons. These results suggest that region-specific DNA methylation and MBDs play an important role in the regulation of differentiation plasticity in neurons.
Neuropilin-1 (NRP1) has been identified as a VEGF-A receptor. DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1. In the present study, the RNA interference of VEGF-A or NRP1 suppressed DJM-1 cell proliferation. Furthermore, the overexpression of the NRP1 wild type restored shNRP1-treated DJM-1 cell proliferation, whereas NRP1 cytoplasmic deletion mutants did not. A co-immunoprecipitation analysis revealed that VEGF-A induced interactions between NRP1 and GIPC1, a scaffold protein, and complex formation between GIPC1 and Syx, a RhoGEF. The knockdown of GIPC1 or Syx reduced active RhoA and DJM-1 cell proliferation without affecting the MAPK or Akt pathway. C3 exoenzyme or Y27632 inhibited the VEGF-A-induced proliferation of DJM-1 cells. Conversely, the overexpression of the constitutively active form of RhoA restored the proliferation of siVEGF-A-treated DJM-1 cells. Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor. A cell-penetrating oligopeptide that targeted GIPC1/Syx complex formation inhibited the VEGF-A-induced activation of RhoA and suppressed DJM-1 cell proliferation. In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.
SHANK3 is a synaptic scaffolding protein enriched in the post-synaptic density of excitatory synapses. Since several SHANK3 mutations have been identified in a particular phenotypic group of patients with autism spectrum disorder (ASD), SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Several SHANK3 isoforms are known to be produced in the developing brain, but they have not been fully investigated. Here, we identified two different amino-terminus truncated Shank3 transcripts. One transcript, designated as Shank3c-3, produces an isoform that contains the entire carboxyl-terminus, but the other transcript, designated as Shank3c-4, produces a carboxyl-terminus truncated isoform. During development, expression of the novel Shank3 transcripts increased after birth, transiently decreased at P14 and then gradually increased again thereafter. We also determined that methyl CpG-binding protein 2 (MeCP2) is involved in regulating expression of the novel Shank3 transcripts. MeCP2 is a transcriptional regulator that has been identified as the causative molecule of Rett syndrome, a neurodevelopmental disorder that includes autistic behavior. We demonstrated a difference between the expression of the novel Shank3 transcripts in wild-type mice and Mecp2-deficient mice. These findings suggest that the SHANK3 isoforms may be implicated in the synaptic abnormality in Rett syndrome. Keywords: autism spectrum disorder, DNA methylation, MeCP2, SHANK3. The SHANK3/proline-rich synapse-associated protein 2 (PROSAP2) gene consists of 22 exons and encodes a multidomain protein that contains ankyrin repeats (ANK) in an amino-terminal region, an Src homology 3 domain, a post-synaptic density 95/discs large/zone occludens-1 domain, a proline-rich region, a homer-binding region, a cortactin-binding region and a sterile alpha motif (SAM) . SHANK3 is abundantly expressed in the heart and moderately expressed in the brain and spleen, and its tissue-specific expression is epigenetically regulated by DNA methylation (Lim et al. 1999;Beri et al. 2007). In the brain, SHANK3 is mainly expressed in neurons, especially in their synapses, and acts as a scaffolding protein in its interactions with various synaptic molecules, including with the NMDA receptor via the post-synaptic density-95 (PSD-95)/guanylate kinase-associated protein complex, with the metabotropic glutamate receptor via homer, and with the GluR1 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (Lim et al. 1999;Naisbitt et al. 1999;Sheng and Kim 2000;Boeckers et al. 2004;Uchino et al. 2006). Address correspondence and reprint requests to Shigeo Uchino, Department of Biosciences, School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan. E-mail: uchino@nasu.bio.teikyo-u.ac.jpAbbreviations used: AMPA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate; ASD, autism spectrum disorder; MeCP2, methyl CpGbinding protein 2; SH3, Src homology 3; PDZ, post-synaptic density 95/ disc...
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