Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells

BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2''-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.     

The all-trans retinoic acid (atRA)-regulated gene Calmin (Clmn) regulates cell cycle exit and neurite outgrowth in murine neuroblastoma (Neuro2a) cells

The vitamin A metabolite all-trans retinoic acid (atRA) functions in nervous system development and regulates cell proliferation and differentiation. Neuroblastoma cells (SH-SY5Y and Neuro2a or N2A) exposed to atRA undergo growth inhibition and neuronal differentiation, both of which are preceded by an increase in Clmn mRNA. Treatment of N2A cells with atRA produces a reduction in phosphohistone 3 immunostaining and BrdU incorporation, both indicators of a reduction in cell proliferation. These effects are nearly eliminated in atRA-treated shClmn knockdown cells. Loss of Clmn in the mouse N2A cell line also results in a significant reduction of atRA-mediated neurite outgrowth, a response that can be rescued by reintroduction of the Clmn sequence. In contrast, ectopic overexpression of Clmn produces an increase in the cyclin dependent kinase inhibitor, p21(Cip1), a decrease in cyclin D1 protein and an increase in hypophosphorylated Rb, showing that Clmn participates in G(1)/S arrest. Clmn overexpression alone is sufficient to inhibit N2A cell proliferation, whereas both Clmn and atRA must be present to induce neurite outgrowth. This study shows that the atRA-responsive gene Clmn promotes exit from the cell cycle, a requisite event for neuronal differentiation.     

Coordination of proliferation and neuronal differentiation by the retinoblastoma protein family

Once neurons enter the post‐mitotic G0 phase during central nervous system (CNS) development, they lose their proliferative potential. When neurons re‐enter the cell cycle during pathological situations such as neurodegeneration, they undergo cell death after S phase progression. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are highly coordinated. In this review, the coordination of cell cycle control and neuronal differentiation during development are discussed, focusing on regulation by the Rb family of tumor suppressors (including p107 and p130), and the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors. Based on recent findings suggesting roles for these families in regulating neurogenesis and neuronal differentiation, I propose that the Rb family is essential for daughter cells of neuronal progenitors to enter the post‐mitotic G0 phase without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family regulates the timing of neuronal progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina. Rb's lack of involvement in regulating the initiation of neuronal differentiation may explain why Rb family‐deficient retinoblastomas characteristically exhibit neuronal features.     

p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina

A precise balance between proliferation and differentiation must be maintained during retinal development to obtain the correct proportion of each of the seven cell types found in the adult tissue. Cyclin kinase inhibitors can regulate cell cycle exit coincident with induction of differentiation programs during development. We have found that the p57(Kip2) cyclin kinase inhibitor is upregulated during G(1)/G(0) in a subset of retinal progenitor cells exiting the cell cycle between embryonic day 14.5 and 16.5 of mouse development. Retroviral mediated overexpression of p57(Kip2) in embryonic retinal progenitor cells led to premature cell cycle exit. Retinae from mice lacking p57(Kip2) exhibited inappropriate S-phase entry and apoptotic nuclei were found in the region where p57(Kip2) is normally expressed. Apoptosis precisely compensated for the inappropriate proliferation in the p57(Kip2)-deficient retinae to preserve the correct proportion of the major retinal cell types. Postnatally, p57(Kip2) was found to be expressed in a novel subpopulation of amacrine interneurons. At this stage, p57(Kip2 )did not regulate proliferation. However, perhaps reflecting its role during this late stage of development, animals lacking p57(Kip2) showed an alteration in amacrine subpopulations. p57(Kip2) is the first gene to be implicated as a regulator of amacrine subtype/subpopulation development. Consequently, we propose that p57(Kip2) has two roles during retinal development, acting first as a cyclin kinase inhibitor in mitotic progenitor cells, and then playing a distinct role in neuronal differentiation.     

Regulatory system for the G1‐arrest during neuronal development in Drosophila

Neuronal network consists of many types of neuron and glial cells. This diversity is guaranteed by the constant cell proliferation of neuronal stem cells following stop cell cycle re‐entry, which leads to differentiation during development. Neuronal differentiation occurs mainly at the specific cell cycle phase, the G1 phase. Therefore, cell cycle exit at the G1 phase is quite an important issue in understanding the process of neuronal cell development. Recent studies have revealed that aberrant S phase re‐entry from the G1 phase often links cellular survival. In this review we discuss the different types of G1 arrest on the process of neuronal development in Drosophila. We also describe the issue that aberrant S phase entry often causes apoptosis, and the same mechanism might contribute to sensory organ defects, such as deafness.     

Lembehyne A, a spongean polyacetylene, induces neuronal differentiation in neuroblastoma cell.

Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.     

Pyridoacridine alkaloids inducing neuronal differentiation in a neuroblastoma cell line,from marine sponge Biemna fortis

A new and three known pyridoacridine alkaloids were isolated from the Indonesian marine sponge Biemna fortis as neuronal differentiation inducers against a murine neuroblastoma cell line, Neuro 2A. The chemical structure of the new compound, labuanine A (1), was determined by spectroscopic study and chemical conversion. These pyridoacridine alkaloids induced multipolar neuritogenesis in more than 50% of cells at 0.03-3 micro M concentration. Compound 3, which showed the strongest neuritogenic activity among them, also induced increase of acetylcholinesterase, a neuronal marker in Neuro 2A and arrested cell cycle at the G2/M phase.     

Lamina-associated polypeptide 2alpha regulates cell cycle progression and differentiation via the retinoblastoma-E2F pathway

Lamina-associated polypeptide (LAP) 2alpha is a nonmembrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP2alpha in fibroblasts reduced proliferation and delayed entry into the cell cycle from a G0 arrest. In contrast, stable down-regulation of LAP2alpha by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2alpha-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2alpha COOH terminus directly bound Rb, and overexpressed LAP2alpha inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2alpha associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2alpha in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP2alpha on cell cycle progression and differentiation may be highly relevant for the cell- and tissue-specific phenotypes observed in laminopathic diseases.     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.     

Confluence-induced cell cycle exit involves pre-mitotic CDK inhibition by p27Kip1 and cyclin D1 downregulation

Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G1, requires pRb family members, and is driven by p27Kip1-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G1, prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G2 phase and requires the Ras-dependent induction of cyclin D1, which promotes following G1/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Over-expression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.     

G1 arrest and differentiation can occur independently of Rb family function

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.     

The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein

Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.