Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: relevance to neuronal differentiation

Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth-associated protein-43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT-PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion-like clusters of neuronal cells induced upon RA exposure. MMP-2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor-alpha (TNFalpha), or phorbol 12-myristate 13-acetate (PMA) treatment. In contrast, MMP-9 was inducible by RA, TNFalpha, or PMA. MMP-9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFalpha or PMA potentiated RA-induced MMP-9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP-9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP-9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP-9 could participate in the neurite growth process and cell migration and organization into ganglion-like clusters.     

Retinoic acid-induced differentiation of human neuroblastoma: a cell variant system showing two distinct responses

Retinoic acid (RA) has been shown to induce the differentiation of human neuroblastoma cells in vitro. In this study, we describe two variants of the SK-N-SH human neuroblastoma cell line that have dramatically different responses to RA. RA induces neuronal-like differentiation characterized by extensive neurite outgrowth, thick neurite bundles, and large cellular aggregates of SK-N-SH-N (SH-N) cells. In contrast, RA treatment of SK-N-SH-F (SH-F) cultures transforms the small neuroblast cells into large flattened, fibroblastic or epithelial-like cells. Karyotype analysis verified that the SH-N and SH-F cultures were derived from a common precursor cell. Confirmation of their markedly different responses to RA was obtained by metabolic labelling of glycoproteins and SDS-PAGE analysis. While both sublines showed very similar Coomassie-labelled protein bands and glycoprotein profiles in control cultures, dramatic differences between the lines were revealed following RA treatment. In contrast to their similar protein profiles, untreated SH-N and SH-F cells had quite different patterns of ganglioside biosynthesis in that GM3 was detected in SH-F cells but not in SH-N, while GM1 was only detected in SH-N. Cellular RA binding protein (CRABP) was detected in both SH-F and SH-N cells and their RA-transformed derivatives. These results demonstrate heterogeneity in the response to RA of neuroblastoma cells derived from a common origin that cannot be accounted for by differences in CRABP content. The SH-N and SH-F neuroblastoma sublines should provide a useful system for further studies of the molecular processes through which RA exerts its differentiation-inducing activity on this type of tumor.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Involvement of membrane protein GDE2 in retinoic acid-induced neurite formation in Neuro2A cells

We show that a glycerophosphodiester phosphodiesterase homolog, GDE2, is widely expressed in brain tissues including primary neurons, and that the expression of GDE2 in neuroblastoma Neuro2A cells is significantly upregulated during neuronal differentiation by retinoic acid (RA) treatment. Stable expression of GDE2 resulted in neurite formation in the absence of RA, and GDE2 accumulated at the regions of perinuclear and growth cones in Neuro2A cells. Furthermore, a loss-of-function of GDE2 in Neuro2A cells by RNAi blocked RA-induced neurite formation. These results demonstrate that GDE2 expression during neuronal differentiation plays an important role for growing neurites.     

Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X₇ receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X₇ receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X₇ receptors. Functional inhibition of P2X₇ receptors by P2X₇ receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X₇ receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were decreased in either RA- or oATP-differentiated or P2X₇ receptor knockdown N2a cells. Simply cultured N2a cells in low Ca²⁺ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X₇ receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X₇ receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X₇ receptor is important in maintaining cell survival of N2a neuroblastoma cells.     

Heterogeneity in retinoic acid signaling in neuroblastomas: Role of matrix metalloproteinases in retinoic acid-induced differentiation

Causes of retinoid resistance often observed in neuroblastomas are unknown. We studied all trans-retinoic acid (RA) signaling in neuroblastoma cells differing in N-myc levels in terms of neurite formation, expression of tissue transglutaminase, neuronal marker proteins, matrix metalloproteinases (MMPs), and activation of Rac1 and Cdc42. Poor invasiveness observed in SH-SY5Y, LA-N-5, and SMS-KCNR cells was associated with RA-induced neurite formation, Cdc42 activation and N-myc down regulation; expression of constitutively active Cdc42 down regulated N-myc expression and reduced invasion in RA-resistant SK-N-BE(2) and IMR32 cells. RA treatment for 24 h transiently increased invasion and expression of MMP9 in SH-SY5Y, LA-N-5 and MMP2 in SMS-KCNR cells. MMP inhibition prevented RA-induced neurite formation indicating a role in differentiation. Variation in RA signaling thus follows a defined pattern and relates to invasive potential. A defective RA signaling might result in retinoid resistance and unpredictable clinical outcome observed in some neuroblastomas.     

Retinoic acid induces neuronal differentiation of embryonal carcinoma cells by reducing proteasome-dependent proteolysis of the cyclin-dependent inhibitor p27.

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.     

Neurite Extension and Increased Expression of R1 Cyclic AMP-Binding Protein in Ouabain-Resistant Neuroblastoma Mutants

Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N-18 mouse neuroblastoma cell line selected for their ouabain-resistant (ouar) property. When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N-18 cells. The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell. This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000-dalton RI cyclic AMP (cAMP)-binding protein. The growth rate, intracellular level of cAMP, and acetylcholinesterase activity of the ouar cell lines were not significantly different from those of the parental N-18 neuroblastoma cells. Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6, O2-dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and RI cAMP-binding activities. The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP-induced differentiated neuroblastoma cell suggests that these two protocols yielded different degrees of differentiation. Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells.     

Extracellular-Regulated Kinases and Phosphatidylinositol 3-Kinase Are Involved in Brain-Derived Neurotrophic Factor-Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH-SY5Y

Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH-SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA-predifferentiated SH-SY5Y cells with brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), or neurotrophin 4/5 (NT-4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal-regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT-3, or NT-4/5, but not NGF, promotes cell survival and neurite outgrowth in serum-free medium. The mitogen-activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF-induced neurite outgrowth and growth-associated protein-43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3-kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.     

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

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.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Induction of K-channel expression in a neuroblastoma cell line

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.