Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.     

Activation of c-Jun-N-terminal kinase is required for apoptosis triggered by glutathione disulfide in neuroblastoma cells

Changes in intracellular redox status are crucial events that trigger downstream proliferation or death responses through activation of specific signaling pathways. Moreover, cell responses to oxidative challenge may depend on the pattern of redox-sensitive molecular factors. The stress-activated protein kinases c-Jun-N-terminal kinase (JNK) and p38 MAP kinase (p38MAPK) are implicated in different forms of apoptotic neuronal cell death. Here, we investigated the effects, on neuroblastoma cells, of the prooxidant molecule GSSG, which we previously demonstrated to be an efficient proapoptotic compound able to activate the p38MAPK death pathway in promonocytic cells. We found that neuroblastoma cells are not prone to GSSG-induced apoptosis, although the treatment slightly induced growth arrest through the accumulation of p53 and its downstream target gene, p21. However, GSSG treatment became cytotoxic when cells were previously depleted of intracellular GSH content. Under this condition, apoptosis was triggered by an increased production of superoxide that led to a specific activation of the JNK-dependent pathway. The involvement of superoxide and JNK was demonstrated by cell death inhibition in experiments carried out in the presence of Cu,Zn superoxide dismutase or with specific inhibitors of JNK activity. Our data give support to the studies that indicate preferential requirements for the involvement of stress-activated kinases in apoptotic neuronal cells.     

Activation of caspases is required for osteoblastic differentiation

Previous studies have shown that mouse osteoblastic MC3T3-E1 cells undergo apoptosis when exposed to a mixture of proinflammatory cytokines. Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e. the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1, as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8, caspase-2, and caspase-3, in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8, caspase-2, and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.     

Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.     

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.     

Synergistic effect of retinoic acid and dehydroepiandrosterone on differentiation of human neuroblastoma cells

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK-N-BE) was evaluated measuring growth rate, motility, neurite extension and GAP-43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor-mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP-2, whereas MMP-9 expression is up-regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co-treatment with the two drugs.     

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.     

Activation of extracellular-signal regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes

Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.     

Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be an useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure, ressembles of those seen in mammalian wound healing. As puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during dorsal closure, we investigated puc gene expression during wound healing in Drosophila. We showed that puc gene expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. We conclude that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration such as wound healing in vertebrates.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Promyelocytic leukemia-nuclear body formation is an early event leading to retinoic acid-induced differentiation of neuroblastoma cells

Neuroblastoma is one of the most common cancers in children. Neuroblastoma differentiation is linked to the presence of the promyelocytic leukemia (PML) protein. Retinoic acid, a powerful differentiation-inducer in vitro , is a potent agent for the treatment of neuroblastoma. Using two different human neuroblastoma cell lines, SH-SY5Y and LA-N-5, we show here that PML protein leads to the formation of nuclear bodies (PML-NB) after only 1 h of retinoic acid treatment and that this formation is mediated by the extracellular signal-regulated kinase (ERK) pathway. Inhibition of protein kinase C also leads to formation of PML-NB via the ERK pathway. Both sumoylation and phosphorylation of PML in an ERK-dependent pathway are also required for formation of PML-NB. Finally, we show that PML-NB formation in neuroblastoma cells is associated with neurite outgrowth. These results support the proposal that the formation of PML-NB is correlated with the differentiation of neuroblastoma cells.     

Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function.

ZAP-70 is a protein tyrosine kinase (PTK) required for T-cell development and T-cell antigen receptor (TCR) function. ZAP-70 is associated with the phosphorylated antigen receptor and undergoes tyrosine phosphorylation following receptor activation. We demonstrate here that tyrosine phosphorylation of ZAP-70 results in an increase in its catalytic activity and that this activation is mediated by the phosphorylation of tyrosine residue 493 by the src family of PTKs. The activity of baculoviral expressed ZAP-70 was up-regulated 10-fold when ZAP-70 was co-infected and phosphorylated by the src family PTK, lck. Mutation of Y493 alone abrogated the ability of ZAP-70 to be activated by lck. Moreover, we demonstrate that phosphorylation of Y493 and activation of ZAP-70 is required for antigen receptor-mediated induction of interleukin-2 (IL-2) secretion in lymphocytes.