HES1 promoter activation dynamics reveal the plasticity, stemness and heterogeneity in neuroblastoma cancer stem cells.

Notch signaling and its downstream gene target HES1 play a critical role in regulating and maintaining cancer stem cells (CSCs), similar to as they do during embryonic development. Here, we report a unique subclass of Notch-independent Hes-1 (NIHes-1)-expressing CSCs in neuroblastoma. These CSCs maintain sustained HES1 expression by activation of HES1 promoter region upstream of classical CBF-1 binding sites, thereby completely bypassing Notch receptor-mediated activation. These stem cells have self-renewal ability and potential to generate tumors. Interestingly, we observed that NIHes-1 CSCs could transition to Notch-dependent Hes-1-expressing (NDHes-1) CSCs where HES1 is expressed by Notch receptor-mediated promoter activation. We observed that NDHes-1-expressing CSCs also had the potential to transition to NIHes-1 CSCs and during this coordinated bidirectional transition, both CSCs gave rise to the majority of the bulk cancer cells, which had an inactive HES1 promoter (PIHes-1). A few of these PIHes-1 cells were capable of reverting into a CSC state. These findings explain the existence of a heterogenic mode of HES1 promoter activation within the IMR-32 neuroblastoma cell line and the potential to switch between them. This article has an associated First Person interview with the first authors of the paper.

Pleiotropic Hes-1 Concomitant with its Differential Activation Mediates Neural Stem Cell Maintenance and Radial Glial Propensity in Developing Neocortex.

Notch signaling pathway and its downstream effector Hes-1 are well known for their role in cortical neurogenesis. Despite the canonical activation of Hes-1 in developing neocortex, recent advances have laid considerable emphasis on Notch/CBF1-independent Hes-1 (NIHes-1) expression with poor understanding of its existence and functional significance. Here, using reporter systems and in utero electroporation, we could qualitatively unravel the existence of NIHes-1 expressing neural stem cells from the cohort of dependent progenitors throughout the mouse neocortical development. Though Hes-1 expression is maintained in neural progenitor territory at all times, a simple shift from Notch-independent to -dependent state makes it pleiotropic as the former maintains the neural stem cells in a non-dividing/slow-dividing state, whereas the latter is very much required for maintenance and proliferation of radial glial cells. Therefore, our results provide an additional complexity in neural progenitor heterogeneity regarding differential Hes-1 expression in the germinal zone during neo-cortical development.

Hes1: the maestro in neurogenesis.

The process of neurogenesis is well orchestrated by the harmony of multiple cues in a spatiotemporal manner. In this review, we focus on how a dynamic gene, Hes1, is involved in neurogenesis with the view of its regulation and functional implications. Initially, we have reviewed the immense functional significance drawn by this maestro during neural development in a context-dependent manner. How this indispensable role of Hes1 in conferring the competency for neural differentiation partly relies on the direct/indirect mode of repression mediated by very specific structural and functional arms of this protein has also been outlined here. We also review the detailed molecular mechanisms behind the well-tuned oscillatory versus sustained expression of this antineurogenic bHLH repressor, which indeed makes it a master gene to implement the elusive task of neural progenitor propensity. Apart from the functional aspects of Hes1, we also discuss the molecular insights into the endogenous regulatory machinery that regulates its expression. Though Hes1 is a classical target of the Notch signaling pathway, we discuss here its differential expression at the molecular, cellular, and/or regional level. Moreover, we describe how its expression is fine-tuned by all possible ways of gene regulation such as epigenetic, transcriptional, post-transcriptional, post-translational, and environmental factors during vertebrate neurogenesis.

Molecular mechanisms in H2O2-induced increase in AT1 receptor gene expression in cardiac fibroblasts: A role for endogenously generated Angiotensin II.

The AT1 receptor (AT1R) mediates the manifold actions of angiotensin II in the cardiovascular system. This study probed the molecular mechanisms that link altered redox status to AT1R expression in cardiac fibroblasts. Real-time PCR and western blot analysis showed that H2O2 enhances AT1R mRNA and protein expression via NADPH oxidase-dependent reactive oxygen species induction. Activation of NF-κB and AP-1, demonstrated by electrophoretic mobility shift assay, abolition of AT1R expression by their inhibitors, Bay-11-7085 and SR11302, respectively, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of AT1R by NF-κB and AP-1 in H2O2-treated cells. Further, inhibition of ERK1/2, p38 MAPK and c-Jun N-terminal kinase (JNK) using chemical inhibitors or by RNA interference attenuated AT1R expression. Inhibition of the MAPKs showed that while ERK1/2 and p38 MAPK suffice for NF-κB activation, all three kinases are required for AP-1 activation. H2O2 also increased collagen type I mRNA and protein expression. Interestingly, the AT1R antagonist, candesartan, attenuated H2O2-stimulated AT1R and collagen mRNA and protein expression, suggesting that H2O2 up-regulates AT1R and collagen expression via local Angiotensin II generation, which was confirmed by real-time PCR and ELISA. To conclude, oxidative stress enhances AT1R gene expression in cardiac fibroblasts by a complex mechanism involving the redox-sensitive transcription factors NF-κB and AP-1 that are activated by the co-ordinated action of ERK1/2, p38 MAPK and JNK. Importantly, by causally linking oxidative stress to Angiotensin II and AT1R up-regulation in cardiac fibroblasts, this study offers a novel perspective on the pathogenesis of cardiovascular diseases associated with oxidative stress.

Molecular basis and functional significance of Angiotensin II-induced increase in Discoidin Domain Receptor 2 gene expression in cardiac fibroblasts.

Delineation of mechanisms underlying the regulation of fibrosis-related genes in the heart is an important clinical goal as cardiac fibrosis is a major cause of myocardial dysfunction. This study probed the regulation of Discoidin Domain Receptor 2 (DDR2) gene expression and the regulatory links between Angiotensin II, DDR2 and collagen in Angiotensin II-stimulated cardiac fibroblasts. Real-time PCR and western blot analyses showed that Angiotensin II enhances DDR2 mRNA and protein expression in rat cardiac fibroblasts via NADPH oxidase-dependent reactive oxygen species induction. NF-κB activation, demonstrated by gel shift assay, abolition of DDR2 expression upon NF-κB inhibition, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of DDR2 by NF-κB in Angiotensin II-treated cells. Inhibitors of Phospholipase C and Protein kinase C prevented Angiotensin II-dependent p38 MAPK phosphorylation that in turn blocked NF-κB activation. Angiotensin II also enhanced collagen gene expression. Importantly, the stimulatory effects of Angiotensin II on DDR2 and collagen were inter-dependent as siRNA-mediated silencing of one abolished the other. Angiotensin II promoted ERK1/2 phosphorylation whose inhibition attenuated Angiotensin II-stimulation of collagen but not DDR2. Furthermore, DDR2 knockdown prevented Angiotensin II-induced ERK1/2 phosphorylation, indicating that DDR2-dependent ERK1/2 activation enhances collagen expression in cells exposed to Angiotensin II. DDR2 knockdown was also associated with compromised wound healing response to Angiotensin II. To conclude, Angiotensin II promotes NF-κB activation that up-regulates DDR2 transcription. A reciprocal regulatory relationship between DDR2 and collagen, involving cross-talk between the GPCR and RTK pathways, is central to Angiotensin II-induced increase in collagen expression in cardiac fibroblasts.

Hes-1 regulates the excitatory fate of neural progenitors through modulation of Tlx3 (HOX11L2) expression.

Tlx3 (HOX11L2) is regarded as one of the selector genes in excitatory versus inhibitory fate specification of neurons in distinct regions of the nervous system. Expression of Tlx3 in a post-mitotic immature neuron favors a glutamatergic over GABAergic fate. The factors that regulate Tlx3 have immense importance in the fate specification of glutamatergic neurons. Here, we have shown that Notch target gene, Hes-1, negatively regulates Tlx3 expression, resulting in decreased generation of glutamatergic neurons. Down-regulation of Hes-1 removed the inhibition on Tlx3 promoter, thus promoting glutamatergic differentiation. Promoter-protein interaction studies with truncated/mutated Hes-1 protein suggested that the co-repressor recruitment mediated through WRPW domain of Hes-1 has contributed to the repressive effect. Our results clearly demonstrate a new and unique role for canonical Notch signaling through Hes-1, in neurotransmitter/subtype fate specification of neurons in addition to its known functional role in proliferation/maintenance of neural progenitors.

Non-canonical activation of Notch signaling/target genes in vertebrates.

Evolutionarily conserved Notch signaling orchestrates diverse physiological mechanisms during metazoan development and homeostasis. Classically, ligand-activated Notch receptors transduce the signaling cascade through the interaction of DNA-bound CBF1-co-repressor complex. However, recent reports have demonstrated execution of a CBF1-independent Notch pathway through signaling cross-talks in various cells/tissues. Here, we have tried to congregate the reports that describe the non-canonical/CBF1-independent Notch signaling and target gene activation in vertebrates with specific emphasis on their functional relevance.

Wnt5a is a crucial regulator of neurogenesis during cerebellum development.

The role of Wnt5a has been extensively explored in various aspects of development but its role in cerebellar development remains elusive. Here, for the first time we unravel the expression pattern and functional significance of Wnt5a in cerebellar development using Wnt5a-/- and Nestin-Cre mediated conditional knockout mouse models. We demonstrate that loss of Wnt5a results in cerebellar hypoplasia and depletion of GABAergic and glutamatergic neurons. Besides, Purkinje cells of the mutants displayed stunted, poorly branched dendritic arbors. Furthermore, we show that the overall reduction is due to decreased radial glial and granule neuron progenitor cell proliferation. At molecular level we provide evidence for non-canonical mode of action of Wnt5a and its regulation over genes associated with progenitor proliferation. Altogether our findings imply that Wnt5a signaling is a crucial regulator of cerebellar development and would aid in better understanding of cerebellar disease pathogenesis caused due to deregulation of Wnt signaling.

Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development.

Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6(-/-)Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD).

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways.

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.