Purpose
Retinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of
RB1
gene. However, other oncogenic mutations such as
MYCN
amplification can induce retinoblastoma ...with proficient
RB1
. Previously, we established
RB1
-proficient
MYCN
-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in
MYCN
-induced retinoblastoma carcinogenesis based on the model in chicken.
Methods
MYCN
transformed retinal cells in culture were obtained from
in vivo MYCN
electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells.
Results
The expression profile of
MYCN
-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of
E2Fs
. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed
RB1
proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis.
Conclusion
In conclusion, with proficient
RB1
,
MYCN
-induced high level of
E2F
expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a
RB1
-deficient tumour.
Purpose: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular ...hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells.
Material and Methods: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques.
Results: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001).
Conclusion: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells.
Abbreviations: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR
Thorough investigation of a neuronal population can help reveal key aspects regarding the nervous system and its development. The retinal horizontal cells have several extraordinary features making ...them particularly interesting for addressing questions regarding fate assignment and subtype specification. In this review we discuss and summarize data concerning the formation and diversity of horizontal cells, how morphology is correlated to molecular markers, and how fate assignment separates the horizontal lineage from the lineages of other retinal cell types. We discuss the novel and unique features of the final cell cycle of horizontal cell progenitors and how they may relate to retinoblastoma carcinogenesis.
Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the ...capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173) EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-inhibitor (AG1478), EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001), consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in which endothelins among several other functions, serve as an injury-signal that regulate the gliotic response of Müller cells.
Studies of neurotrophins and Trk receptors in jawless fish have shed light on the course of events underlying the formation of these gene families. They evolved early in vertebrate history during ...major gene duplication events, before the appearance of cartilaginous fish. The existence of multiple genes has permitted the diversification of neurotrophin and Trk receptor expression, and thereby enabling the acquisition of specific functions in selective neuronal populations.
GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the ...effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca(2+) in regulating the cell cycle.
Retinal injury induces Müller cell dedifferentiation by activating extracellular signal-regulated kinase (ERK) signaling. Stimulation of α2-adrenergic receptors protects against injury but also ...activates ERK in Müller cells. The purpose of this work was to study the effect of α2-adrenergic signaling on injury-induced ERK and Müller cell dedifferentiation. We tested the hypothesis that α2-stimulation triggers negative feedback regulation of the injury-induced ERK pathway that attenuates Müller cell dedifferentiation.
Chicken retina injured by N-methyl-D-aspartate and cultured primary Müller cells were stimulated by the α2-adrenergic agonist brimonidine. Immunostaining, quantitative RT-PCR, and Western blot techniques in combination with receptor blockers were used for analysis of the cellular responses.
Alpha2-adrenergic receptor stimulation attenuated injury-induced ERK activation and dedifferentiation of Müller cells as seen by decreased phospho-ERK, expression of transitin, and retinal progenitor cell genes. The attenuation was concomitant with a synergistic upregulation of several negative ERK-signal feedback regulators including ERK-phosphatases, Raf1-, and growth factor receptor-binding proteins. The results were also seen in cultures of primary Müller cells.
Alpha2-adrenergic signaling on Müller cells elicits an intracellular attenuation of the injury response that comprises negative ERK-signaling feedback leading to attenuated Müller cell dedifferentiation. The implications of this study are that adrenergic stress signals may directly modulate glial function in retina and that α2-adrenergic receptor pharmacology may be used to control glial injury response.
Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in ...the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors.
A 208 bp gene regulatory sequence from the chicken
(RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures.
Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with
expression in the early retina.
In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the
regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.
Cell migration plays an important role during the development of the retina. In this work we have studied the migration of newborn horizontal cells in avian embryonic retina. Using the pattern of the ...early expressed transcription factors Lim1 and Prox1 we have shown that horizontal cells migrate bi-directionally from their site of birth, close to the ventricular side, to the adjacent (vitreal) side of the neuroepithelium, where they align just next to the prospective ganglion cell layer before migrating back again to their final laminar position in the external part of the inner nuclear layer. The migration occurs between Hamburger and Hamilton stages 24 and 33, which is equivalent to embryonic day 4.5 and 8. Between stages 26 and 30 the horizontal cells reside close to the ganglion cell layer and intra ocular injections of a cytochalasin D, an actin polymerisation blocker that inhibit migration, at stage 29 interfered with the migration of the horizontal cells to their final destination. Furthermore, using biolistic gene transfer with a green fluorescence protein expression vector of retinal slices we were able to record ventricle-directed migration by time-lapse microscopy. Combining biolistics with immunohistochemistry we showed that transfected cells, which have also been translocated in a ventricular direction were positive for the horizontal cell markers Lim1 and Prox1. The alternative path of migration that is described in this work differs from the generally accepted one for horizontal cells and this knowledge will influence the view of how the molecular determination of horizontal cells is specified.
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