Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Retinoic Acid Is Required for Neural Differentiation of SH-SY5Y Human Neuroblastoma Cells

Abstract

Retinoic acid (RA) induces neural differentiation of SH-SY5Y neuroblastoma cells. We show that the mRNA levels of the differentiation-inhibiting basic helix-loop-helix transcription factors ID1,ID2, and ID3 are down-regulated during RA-induced differentiation of SH-SY5Y cells. The levels of ID proteins decreased in parallel to the observed transcriptional repression. The expression of other basic helix-loop-helix genes changed during RA-induced differentiation: expression of neuroblast-specificASCL1 (HASH-1) gene was promptly reduced after RA treatment, whereas expression of differentiation-promoting genes NEUROD6 (NEX-1, HATH-2) andNEUROD1 was increased. Treatments with 12-O-tetradecanoylphorbol-13-acetate, another inducer of neuroblastoma cell differentiation, also resulted in coordinated down-regulation of ID gene expression, underscoring the role of ID genes in differentiation. Down-regulation ofID gene expression by RA involves a complex mechanism because full transcriptional repression required newly synthesized proteins and signaling by phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, resulting in increased PI3K activity in extracts from RA-treated cells and a rapid increase in phosphorylation of Akt in Ser-473. Inhibition of PI3K by LY294002 impaired RA-induced differentiation, as assessed by morphological and biochemical criteria. We propose that RA, by activating the PI3K/Akt signaling pathway, plays an important role in the regulation of neuronal cell survival. Retinoic acid (RA) induces neural differentiation of SH-SY5Y neuroblastoma cells. We show that the mRNA levels of the differentiation-inhibiting basic helix-loop-helix transcription factors ID1,ID2, and ID3 are down-regulated during RA-induced differentiation of SH-SY5Y cells. The levels of ID proteins decreased in parallel to the observed transcriptional repression. The expression of other basic helix-loop-helix genes changed during RA-induced differentiation: expression of neuroblast-specificASCL1 (HASH-1) gene was promptly reduced after RA treatment, whereas expression of differentiation-promoting genes NEUROD6 (NEX-1, HATH-2) andNEUROD1 was increased. Treatments with 12-O-tetradecanoylphorbol-13-acetate, another inducer of neuroblastoma cell differentiation, also resulted in coordinated down-regulation of ID gene expression, underscoring the role of ID genes in differentiation. Down-regulation ofID gene expression by RA involves a complex mechanism because full transcriptional repression required newly synthesized proteins and signaling by phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, resulting in increased PI3K activity in extracts from RA-treated cells and a rapid increase in phosphorylation of Akt in Ser-473. Inhibition of PI3K by LY294002 impaired RA-induced differentiation, as assessed by morphological and biochemical criteria. We propose that RA, by activating the PI3K/Akt signaling pathway, plays an important role in the regulation of neuronal cell survival. all-trans retinoic acid basic domain helix-loop-helix transcription factor phosphate-buffered saline phosphatidylinositol 3-kinase retinoic acid receptor 9-cis retinoic acid receptor 12-O-tetradecanoylphorbol-13-acetate Cell differentiation is a complex process regulated by an interplay among intrinsic cellular programs, cell-cell and cell-substrate interactions, and a plethora of soluble extracellular signaling molecules, including hormones, growth factors, cytokines, trophic factors, and morphogens. Retinoic acid (RA),1 the biologically active form of vitamin A, plays an important role in early embryonic development and in the generation of several organs and systems, as is the case with the nervous system (for review, see Refs. 1Means A.L. Gudas L.J. Annu. Rev. Biochem. 1995; 64: 201-233Crossref PubMed Scopus (268) Google Scholar, 2Zile M.H. J. Nutr. 1998; 128: 455S-458SCrossref PubMed Google Scholar, 3Ross S.A. McCaffery P.J. Drager U.C. De Luca L.M. Physiol. Rev. 2000; 80: 1021-1054Crossref PubMed Scopus (734) Google Scholar). In vitro, RA also plays a prominent role in regulating the transition from the proliferating precursor cell to the postmitotic differentiated cell, and there are many examples in the literature of distinct cell types whose differentiation is under the control of RA (4Strickland S. Mahdavi V. Cell. 1978; 15: 393-403Abstract Full Text PDF PubMed Scopus (1132) Google Scholar, 5Breitman T.R. Selonick S.E. Collins S.J. Proc. Natl. Acad. Sci. U. S. 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Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar and 13Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1165) Google Scholar). To elucidate the molecular mechanisms through which RA induces differentiation of neural cells, we have analyzed the changes in gene expression associated with RA-induced differentiation using human neuroblastoma cells as a model system. Neuroblastoma is a childhood tumor that arises from neural crest neuroectodermal cells (for review, see Refs. 14Schor N.F. J. Neurooncol. 1999; 41: 159-166Crossref PubMed Scopus (40) Google Scholar and 15Alexander F. Urol. Clin. North Am. 2000; 27 (, vii): 383-392Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Despite their tumoral origin, neuroblastoma cell lines can be induced to differentiate in vitro by several agents, including RA (7Sidell N. J. Natl. Cancer Inst. 1982; 68: 589-596PubMed Google Scholar,16Påhlman S. Odelstad L. Larsson E. Grotte G. Nilsson K. Int. J. Cancer. 1981; 28: 583-589Crossref PubMed Scopus (225) Google Scholar, 17Påhlman S. Meyerson G. Lindgren E. Schalling M. Johansson I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9994-9998Crossref PubMed Scopus (121) Google Scholar, 18Lavenius E. Parrow V. Nånberg E. Påhlman S. Growth Factors. 1994; 10: 29-39Crossref PubMed Scopus (70) Google Scholar). In vitro differentiated neuroblastoma cells have a neuronal phenotype, as judged by their morphology and the expression of biochemical and functional neuronal markers. Therefore, neuroblastoma cells could be considered a useful model system to study the initial phases of neuronal differentiation (19Påhlman S. Hoehner J.C. Nånberg E. Hedborg F. Fagerström S. Gestblom C. Johansson I. Larsson U. Lavenius E. Ortoft E. Eur. J. Cancer. 1995; 31: 453-458Abstract Full Text PDF Scopus (131) Google Scholar). RA derivatives are being employed in the therapy of neuroblastoma, and neuroblastoma patients treated with RA have increased survival rates (20Matthay K.K. Villablanca J.G. Seeger R.C. Stram D.O. Harris R.E. Ramsay N.K. Swift P. Shimada H. Black C.T. Brodeur G.M. Gerbing R.B. Reynolds C.P. N. Engl. J. Med. 1999; 341: 1165-1173Crossref PubMed Scopus (1570) Google Scholar, 21Reynolds C.P. Curr. Oncol. Rep. 2000; 2: 511-518Crossref PubMed Scopus (96) Google Scholar). Nevertheless, little is known about the molecular basis of the therapeutic effects of RA on neuroblastoma tumors. Here we show that RA treatment of SH-SY5Y human neuroblastoma cells leads to changes in the expression levels of bHLH transcription factors, a gene family involved in the regulation of lineage commitment and cell differentiation in many cell types (for review, see Ref. 22Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1376) Google Scholar). The products of ID genes act as dominant-negative inhibitors of differentiation by avoiding the binding of other bHLH transcription factors to their cognate RA treatment of SH-SY5Y neuroblastoma cells leads to a coordinated down-regulation of the differentiation-inhibiting In the expression of the bHLH transcription factor is and the mRNA levels of the differentiation-promoting bHLH factors NEUROD6 andNEUROD1 are increased. These the that RA treatment differentiation by the differentiation-inhibiting and differentiation-promoting bHLH The mechanism through which RA ID gene expression to be complex because repression required newly synthesized proteins and the activity of phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, which is a neuroblastoma cell differentiation, because treatment with LY294002 impaired RA-induced differentiation. The show of of RA to the of neural cell differentiation. In the of the PI3K/Akt signaling by RA a role of RA in the regulation of survival in neural cells, which a cell differentiation and cell survival. SH-SY5Y human neuroblastoma cells in with and in a with The was and the cells RA retinoic and LY294002 from in and to the the of the from from G. C. M. PubMed Scopus Google Scholar). The ID the are and The of a the human The H. A. P. A. PubMed Scopus Google Scholar). The of a the human S. R.C. J. PubMed Scopus Google Scholar). and by from from SH-SY5Y cells. was a including M.H. J.E. R.E. J. Clin. 77: PubMed Scopus Google Scholar). was a of J. K. Proc. Natl. Acad. Sci. U. S. A. 1978; PubMed Scopus Google Scholar). The was a including of the E. J. PubMed Scopus Google Scholar). was from cell by the of and P. N. Biochem. PubMed Scopus Google Scholar). was through and to specific activity with specific activity by was to and G.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: PubMed Scopus Google Scholar). with and to with in and with by and in the of in and of and of was with and by to with the of a and the was to the by with of transcription of of specific activity of and of in nuclear was with of and the was to and P. N. Biochem. PubMed Scopus Google Scholar). The in the was with to the and the was in The was by an in a are in of in a of in of with of and on The was with of and to the and with the to with Cell with and in The was by and the was with a of extracts to to PubMed Scopus Google Scholar), and the proteins to was with and was with the in the with and the was with in and development with was as by the to in the the of and ID3 from Akt was from Cell from and Cell in and cells in and The was by and the was with the to the in the was the is in The was by the with and The extracts in of of of and of the a the a in and the was the was to Cell with and cells in and by The extracts by and was by of cell in to of of in and of of specific control and by of with and the was a with in was in and the of the to with of the was with To the molecular mechanisms through which RA differentiation in SH-SY5Y cells, we analyzed genes using the M. N. D. E. S. PubMed Scopus Google Scholar). several was to the of human ID3 The in the expression of ID3 in cells which was after of treatment with RA after of RA treatment the down-regulation of ID3 mRNA during RA-induced differentiation and nuclear transcription a of the transcription of the ID3 gene the observed RA-induced down-regulation of the ID gene of bHLH factors have involved in the regulation of differentiation in many cell types J. Cell Sci. 2000; PubMed Google Scholar, Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar), we other ID genes are in SH-SY5Y neuroblastoma cells and RA treatment could their expression in was in SH-SY5Y cells levels with of mRNA levels to be because was after of the mRNA was in SH-SY5Y cells. of SH-SY5Y cells with RA resulted in down-regulation and expression, in parallel to the observed RA treatment to a coordinated down-regulation of ID The levels of and ID3 proteins in SH-SY5Y cells reduced during RA treatment, in parallel to the observed changes in mRNA expression, as by with specific ID proteins to and avoiding complex with bHLH proteins and binding to of SH-SY5Y cells with RA resulted in an increase of the to an in with the observed in the of ID We have analyzed the expression of bHLH genes involved in neuronal commitment and differentiation during the of RA-induced neuronal differentiation of SH-SY5Y cells. in the mRNA levels of a gene involved in of the neuronal in proliferating C. A. I. Ortoft E. Larsson C. H. P. Påhlman S. 1999; Google Scholar), reduced after RA the other expression of of the of bHLH factors was The mRNA levels (NEX-1, HATH-2) are and increased after RA of induced by The other bHLH factors and a neuronal differentiation and gene we have the expression of of in cells and increased during RA-induced differentiation, expression after of In differentiation induced by RA to changes in the expression of bHLH transcription factors in SH-SY5Y cells. In to down-regulation of ID RA treatment the transcription of a and the expression of genes from NEUROD6 and In the transcription of a gene as is are that have to neural differentiation in several neuroblastoma cell lines S. Odelstad L. Larsson E. Grotte G. Nilsson K. Int. J. Cancer. 1981; 28: 583-589Crossref PubMed Scopus (225) Google Scholar). We have the expression levels of the ID genes are during differentiation of SH-SY5Y cells. in the levels of and are after of treatment with reduced and of differentiation, with to that observed with RA In induced levels of could be after of treatment reduced during differentiation. The expression of the is down-regulated during differentiation induced by as RA, a through a nuclear hormone and an of underscoring the of the down-regulation of differentiation of neuroblastoma cells. we the molecular mechanisms of RA-induced coordinated down-regulation of The that down-regulation of the expression and after of RA, with a with to other down-regulated genes as in of a regulation of the transcription ofID genes by In RA and that have effects on gene have effects on the expression of the ID an transcriptional regulation of SH-SY5Y cells with an of down-regulation and ID3 expression by RA, transcriptional of genes by RA, the that newly synthesized proteins are required repression of ID genes by the PI3K signaling involved in differentiation of many cell we have the of a specific of on RA-induced repression of ID gene in treatment with LY294002 RA-induced full down-regulation of ID3 gene expression in SH-SY5Y cells, the of other genes by RA These that repression of ID genes by RA a signaling RA treatment of SH-SY5Y cells resulted in of PI3K as by the in vitro with cellular extracts with RA resulted in a increase in PI3K which to increase was the was in the of LY294002 increase of PI3K activity with an increase in the levels of the of the as by with specific increase to be a of an of RA on the of the because a parallel increase in mRNA was Akt is of the of of SH-SY5Y neuroblastoma cells with RA resulted in rapid phosphorylation of the Akt in Akt was as as after of RA treatment and increased to a after of RA The of phosphorylation by RA treatment was with that observed by treatment a known inducer of Akt phosphorylation in SH-SY5Y cells J. 2000; PubMed Scopus Google Scholar). we the of PI3K by specific LY294002 RA-induced differentiation. was by the of cells and by the transcriptional of the RA-induced morphological differentiation, by an increase in the of cells was by the of LY294002 of the cells with the PI3K resulted in a increase in mRNA transcriptional of by RA was by treatment with LY294002 of the gene was also impaired by treatment with LY294002 of is during RA-induced differentiation. a increase of mRNA could be observed in cells. the RA-induced of that of PI3K/Akt signaling by RA is required RA-induced cellular differentiation and the transcription genes involved in the regulation of cell survival. The of the bHLH family of transcription factors are involved in the control of differentiation, and development of many and including the nervous system (for review, see Refs. 22Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1376) Google Scholar, S. Curr. Dev. 7: PubMed Scopus Google Scholar, and J.E. Curr. 7: PubMed Scopus Google Scholar). control is the of the differentiation-promoting and differentiation-inhibiting bHLH ID genes dominant-negative bHLH proteins that are to differentiation by the of differentiation-promoting bHLH proteins (for review, see Refs. J. Cell Sci. 2000; PubMed Google Scholar Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). 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Villablanca J.G. Seeger R.C. Stram D.O. Harris R.E. Ramsay N.K. Swift P. Shimada H. Black C.T. Brodeur G.M. Gerbing R.B. Reynolds C.P. N. Engl. J. Med. 1999; 341: 1165-1173Crossref PubMed Scopus (1570) Google Scholar, 21Reynolds C.P. Curr. Oncol. Rep. 2000; 2: 511-518Crossref PubMed Scopus (96) Google Scholar), and other types (for review, see Ref. E. 2000; 5: PubMed Scopus Google Scholar). from the is that of the PI3K/Akt signaling by RA to an increase of the to cell could be an could be important RA treatment be with on of cell as be the generation of the to the PI3K/Akt pathway, which could have in We are to and to V. A. A. C. E. C. J. L. J. M. A. A. V. F. and A. of and of We the and the