TRPV1 expression and activity during retinoic acid-induced neuronal differentiation

The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca²⁺-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton re-organisation and in neuronal guidance. To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using all-trans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca²⁺ concentration by 30% as well as the relative capsaicin-induced Ca²⁺ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca²⁺ homeostasis.     

Expression of retinoid receptors during the retinoic acid-induced neuronal differentiation of human embryonal carcinoma cells

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.     

A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid-induced neuronal differentiation

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase-AKT.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

Translocation and phosphorylation of calcyclin binding protein during retinoic acid-induced neuronal differentiation of neuroblastoma SH-SY5Y cells

For better understanding of functions of the Calcyclin Binding Protein (CacyBP) and exploring its possible roles in neuronal differentiation, the subcellular localization of human CacyBP was examined in retinoic acid(RA)-induced and uninduced neuroblastoma SH-SY5Y cells. Immunostaining indicated that CacyBP was present in the cytoplasm of uninduced SH-SY5Y cells, in which the resting Ca(2+) concentration was relatively lower than that of RA-induced cells. After the RA induction, immunostaining was seen in both the nucleus and cytoplasm. In the RA-induced differentiated SH-SY5Y cells, CacyBP was phosphorylated on serine residue(s), while it existed in a dephosphorylated form in normal (uninduced) cells. Thus, the phosphorylation of CacyBP occurs when it is translocated to the nuclear region. The translocation of CacyBP during the RA-induced differentiation of SH-SY5Y cells suggested that this protein might play a role in neuronal differentiation.     

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.     

Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells

Background

Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson''s disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/Principal Findings

In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/Significance

Parkinson''s disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.     

Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.     

Phosphatidylinositol transfer protein in murine embryonal carcinoma cells during retinoic acid-induced differentiation

Phosphatidylinositol transfer protein (PI-TP) was studied in P19 embryonal carcinoma (EC) cells at different stages of retinoic acid (RA) induced differentiation. Western blot analysis indicated an increased expression of PI-TP (35 kDa) during differentiation. Western blots of isoelectric focusing gels showed that the 35 kDa band consisted of the PI-carrying form of PI-TP (pl 5.5) and of a novel, more acidic form of PI-TP (pl 5.4), levels of both of which increased during differentiation. These increased levels were not reflected in the in vitro PI-transfer activity of the cytosolic fraction nor in the mRNA levels as analyzed by northern blotting. By using indirect immunofluorescence it was shown that PI-TP is localized in the cytoplasm and associated with perinuclear Golgi structures and that this distribution is slightly affected during RA-induced differentiation. Immunoprecipitation of PI-TP from [³²P]P_i labeled cells demonstrated that the level of phosphorylation of PI-TP is high in undifferentiated P19 EC cells and low after 5 days of RA-induced differentiation. These results strongly suggest that changes in the levels of PI-TP are intimately connected with changes in the growth characteristics of P19 EC cells during RA-induced differentiation. It remains to be established to what extent this connection is governed by the recent finding that PI-TP is an essential cytosolic factor in stimulating phospholipase C activity.     

Modulation of lysosomal-associated membrane glycoproteins during retinoic acid-induced embryonal carcinoma cell differentiation

Differentiation of the murine embryonal carcinoma (EC) cell lines F-9 and PC-13, induced by beta-all transretinoic acid (RA) resulted in an increased level of two lysosomal-associated membrane glycoproteins (LAMP-1 and LAMP-2). After differentiation, the levels of both LAMPs in the EC cells were comparable to those found in visceral and parietal endoderm cell lines (PSA-5E and PYS-2, respectively). RA treatment of the EC cells also resulted in an increase in the apparent Mr of both LAMPs apparently due to increased glycosylation because the deglycosylated LAMP-1 from undifferentiated and from differentiated cells had a similar electrophoretic migration. Indeed, the binding of 125I-labeled L-phytohemagglutinin (L-PHA) to glycoproteins with Mr or 90,000-130,000 increased after differentiation and about 24 times more 125I-labeled L-PHA bound to LAMP-1 isolated by immunoprecipitation from extracts of RA-treated F-9 cells than to LAMP-1 from undifferentiated cells. The increased level of the LAMPs was detected in F-9 cells treated with greater than 10(-7) M RA and required greater than 48 h of treatment as did the increased expression of the B1 chain of laminin, an established marker for differentiation in this system. LAMP-1- and L-PHA-reactive glycoproteins were localized by fluorescence techniques to intracellular vesicles, presumably lysosomes, and to the cell surface and both increased after RA treatment. LAMP-2 was barely detectable intracellularly in undifferentiated cells but could be detected clearly after differentiation. In contrast, no LAMP-2 could be detected on the cell surface either before or after differentiation of F-9 cells. The increased level and glycosylation of both LAMP-1 and LAMP-2 was observed also in cells treated with a synthetic chalcone carboxylic acid analog of RA and by combination of either retinoid with dibutyryl cyclic AMP. These results demonstrate that differentiation of EC cells is accompanied by changes in the synthesis and glycosylation of LAMP glycoproteins and that these changes are specific for the cell type that results after differentiation.