The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.     

S100B increases proliferation in PC12 neuronal cells and reduces their responsiveness to nerve growth factor via Akt activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner.     

Expression profile identifies novel genes involved in neuronal differentiation

In the presence of NGF, PC12 cells extend neuronal processes, cease cell division, become electrically excitable, and undergo several biochemical changes that are detectable in developing sympathetic neurons. We investigated the expression pattern of the apoptosis-related genes at each stage of neuronal differentiation using a cDNA microarray containing 320 apoptosis-related rat genes. By comparing the expression patterns through time-series analysis, we identified candidate genes that appear to regulate neuronal differentiation. Among the candidate genes, HO2 was selected by real-time PCR and Western blot analysis. To identify the roles of selected genes in the stages of neuronal differentiation, transfection of HO2 siRNA in PC12 cells was performed. Down-regulation of HO2 expression causes a reduction in neuronal differentiation in PC12 cells. Our results suggest that the HO2 gene could be related to the regulation of neuronal differentiation levels.     

Expression of the retinoblastoma-related p107 and Rb2/p130 genes in human placenta: an immunohistochemical study

It has been proposed that tumor suppressor genes may have a role in the mechanisms of proliferation and differentiation during human placental development. The Retinoblastoma gene family is a well known family of tumor suppressor genes. Many studies have pointed out a role of this family not only in cell cycle progression, but also during development and differentiation. On the light of these observations we have investigated the immunohistochemical expression pattern of the Retinoblastoma family members, p107 and Rb2/p130 in human placenta samples in first trimester and full-term placental sections. p107 and pRb2/p130 showed the most abundant expression levels during the first trimester of gestation and progressively declined to being barely detectable in the placenta by late gestation. These results indicate that the expression of the above genes is modulated during placental development and suggest a mechanism for controlling trophoblast proliferation.     

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in 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.     

Dominant Negative ATF1 Blocks Cyclic AMP-Induced Neurite Outgrowth in PC12D Cells

Abstract: Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.     

Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells.     

Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation.