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B-ind1, a Novel Mediator of Rac1 Signaling Cloned from Sodium Butyrate-treated Fibroblasts*

  • Delphine Courilleau
    Footnotes
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Eric Chastre
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Michèle Sabbah
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Gérard Redeuilh
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Azeddine Atfi
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Jan Mester
    Correspondence
    To whom correspondence should be addressed. Tel.: 33-1-49-28-46-14; Fax: 33-1-44-74-93-18
    Affiliations
    INSERM U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France
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  • Author Footnotes
    * This work was supported in part by INSERM and Association pour la Recherche sur le Cancer Grant 9835.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Z97207 and AJ271091.
    ‡ Supported by doctoral grants from the Ministère de l'Enseignement Supérieur et de la Recherche and the Association pour la Recherche sur le Cancer.
Open AccessPublished:June 09, 2000DOI:https://doi.org/10.1074/jbc.M000887200
      Sodium butyrate is a multifunctional agent known to inhibit cell proliferation and to induce differentiation by modulating transcription. We have performed differential display analysis to identify transcriptional targets of sodium butyrate in Balb/c BP-A31 mouse fibroblasts. A novel butyrate-induced transcript B-ind1 has been cloned by this approach. The human homologue of this transcript contains an open reading frame that codes for a protein of 370 amino acids without known functional motifs. In transfected cells, the B-ind1 protein has been found to potentiate different effects of the small GTPase Rac1, such as c-Jun N-terminal kinase activation and transcriptional activity of nuclear factor κB (NF-κB). In addition, we have demonstrated that B-ind1 forms complexes with the constitutively activated Rac1 protein. To investigate the role of B-ind1 in Rac1 signaling, we have constructed several deletion mutants of B-ind1 and tested their ability to affect the activation of NF-κB by Rac1. Interestingly, the fragment encoding the median region of human B-ind1 acted as a dominant-negative variant to block Rac1-mediated NF-κB activity. These data define B-ind1 as a novel component of Rac1-signaling pathways leading to the modulation of gene expression.
      HDAC
      histone deacetylase
      NF-κB
      nuclear factor κB
      JNK
      c-Jun N-terminal kinase
      GST
      glutathione S-transferase
      PCR
      polymerase chain reaction
      MOPS
      4-morpholinepropanesulfonic acid
      hB-ind1
      human B-ind1
      Sodium butyrate is a multifunctional agent known as an efficient inhibitor of cell proliferation, an inducer of differentiation in numerous cell lines and apoptosis in certain cell types (
      • Heerdt B.G.
      • Houston M.A.
      • Augenlicht L.H.
      ,
      • Janson W.
      • Brandner G.
      • Siegel J.
      ). Sodium butyrate exerts most of its biological effects through a reversible inhibition of histone deacetylases (HDAC),1 resulting in histone hyperacetylation and in selective changes in gene expression. Effects similar to those of sodium butyrate are also induced by structurally unrelated inhibitors of HDAC such as trichostatin A (
      • Yoshida M.
      • Kijima M.
      • Akita M.
      • Beppu T.
      ). The acetylation state of core histones plays a critical role in the regulation of transcription. In addition, a DNA butyrate-response element conferring the ability of sodium butyrate to induce gene expression has been identified (
      • Yeivin A.
      • Tang D-C.
      • Taylor M.W.
      ,
      • Gill R.K.
      • Christakos S.
      ). However, sodium butyrate also inhibits the expression of several genes including c-myc (
      • Heruth D.P.
      • Zirnstein G.W.
      • Bradley J.F.
      • Rothberg P.G.
      ) and cyclin D1 (
      • Lallemand F.
      • Courilleau D.
      • Sabbah M.
      • Redeuilh G.
      • Mester J.
      ). p21WAF1, a cellular inhibitor of cyclin-dependent kinase and of the progression in G1 phase of the cell cycle, is induced by sodium butyrate as well as by trichostatin A (
      • Steinman R.A.
      • Hoffman B.
      • Iro A.
      • Guillouf C.
      • Liebermann D.A.
      • El-Houseini M.E.
      ,
      • Archer S.Y.
      • Meng S.
      • Shei A.
      • Hodin R.A.
      ). We have previously reported that in the mouse fibroblast cell line BP-A31 sodium butyrate inhibits the hyperphosphorylation of the tumor suppressor retinoblastoma protein via a transcription-dependent mechanism (
      • Buquet-Fagot C.
      • Lallemand F.
      • Charollais R-H.
      • Mester J.
      ). However, sodium butyrate induces only a small increase in the level of p21WAF1 transcript in this cell line, suggesting that other butyrate-induced gene products may be involved in its antiproliferative action.
      Van Lint et al. (
      • Van Lint C.
      • Emiliani S.
      • Verdin E.
      ) showed that only a small subset of gene promoters is sensitive to inhibition of histone deacetylation by trichostatin A. Thus, cloning of transcripts induced by inhibitors of HDAC may facilitate the identification of effectors responsible for the antiproliferative and/or differentiating actions of this class of drugs. Using the differential display technique (
      • Liang P.
      • Pardee A.B.
      ), we cloned the cDNA of a new gene transcript induced by sodium butyrate in the mouse fibroblast cell line BP-A31. The protein encoded by this transcript (designated B-ind1 for butyrate-induced 1) does not affect cell proliferation and apoptosis but does appear to participate in the control of the transcriptional activity of NF-κB that is stimulated by sodium butyrate. In this context, we show that B-ind1 acts downstream of the small GTPase Rac1 in the biochemical pathway leading to NF-κB activation. B-ind1 also collaborates with the activated form of Rac1 to induce c-Jun N-terminal kinase (JNK) activity. Taken together, these data implicate B-ind1 as a novel component of the sodium butyrate and Rac1-signaling pathways that influence the regulation of gene expression.

      DISCUSSION

      There is strong evidence that the biological effects of HDAC inhibitors are mediated through their ability to directly induce or inhibit the expression of several genes. Our purpose was to search for such genes and study the cellular functions of the corresponding products. Analyzing the differential display PCR patterns obtained with 12 primer combinations, we found two transcripts whose induction by sodium butyrate in the mouse fibroblast cell line BP-A31 was confirmed by Northern blotting. One of these transcripts, B-ind1, was present in quiescent, nontreated cells and was induced by butyrate in a protein synthesis-independent manner. Analysis of the complete cDNA obtained by screening of a human brain cDNA library revealed an open reading frame coding for a protein of 370 amino acids.
      One important finding of our study is the demonstration that hB-ind1 is involved in the signaling pathway mediated by the small GTPase Rac1. In particular, hB-ind1 potentiated the Rac1-induced transcription and JNK activation. Moreover, the median region comprising amino acids 101–277 acted as a dominant-negative variant to inhibit the induction of the NF-κB-dependent promoter by Rac1. The mechanism of action of hB-ind1 may rely on the formation of complexes between Rac1 and its targets. Although the sequence of hB-ind1 reveals no Cdc42/Rac-interactive binding site, a distinctive domain contained in several previously identified Rac1 targets (
      • Burbelo P.D.
      • Drechsel D.
      • Hall A.
      ), the existence of protein complexes containing Rac1 and hB-ind1 was evidenced by coimmunoprecipitation experiments. Because other proteins without the Cdc42/Rac-interactive binding site associate with Rac1 (
      • Tapon N.
      • Nagata K-I.
      • Lamarche N.
      • Hall A.
      ), it is possible that hB-ind1 favors the maintenance of Rac1 complexes with its effector systems. This hypothesis is substantiated by the inhibition of the Rac signaling pathway by the median fragment M of hB-ind1, although this fragment did not form stable complexes with Rac1. The M fragment may act as a dominant-negative variant by competing with endogenous wild-type B-ind1 to sequester downstream targets required in Rac1 signaling.
      In addition, the hB-ind1 protein displays homologies with p23 and related proteins from other species including chicken, yeast, andC. elegans. This suggests that hB-ind1 may be a member of a large family of proteins. Human p23 was first identified as a protein associated with the progesterone receptor (
      • Smith D.F.
      • Faber L.E.
      • Toft D.O.
      ) and was later shown to be in fact a heat shock (Hsp90)-binding protein (
      • Johnson J.L.
      • Beito T.G.
      • Krco C.J.
      • Toft D.O.
      ,
      • Sullivan W.
      • Stensgard B.
      • Caucutt G.
      • Bartha B.
      • McMahon N.
      • Alnemri E.S.
      • Litwack G.
      • Toft D.O.
      ). Both p23 and its yeast homolog Sba1 (
      • Fang Y.
      • Fliss A.E.
      • Rao J.
      • Caplan A.J.
      ) appear to act as Hsp90 cochaperones, but each possesses a chaperone activity of its own (
      • Bose S.
      • Weikl T.
      • Bügl H.
      • Buchner J.
      ). p23 has been postulated to crystallize as a homodimer.
      D. Toft, personal communication.
      These observations lead us to speculate that hB-ind1 may form homodimers with a chaperone type activity toward Rac1. In that case, the dominant-negative action of the M fragment would result from the formation of M/wild-type B-ind1 dimers, depleting Rac1 of its chaperone.
      The role of B-ind1 in the cellular effects of sodium butyrate is not clear. The B-ind1 transcript was present in all tissues examined; its induction by sodium butyrate in cultured cells is not an universal process, and its overexpression in transfected COS-7 cells did not induce cell growth arrest or apoptosis. In the transformed mouse embryo fibroblasts, the cell in which this transcript has been cloned, B-ind1 may participate in the initial differentiation program.
      In conclusion, the strategy of cloning transcripts induced by HDAC inhibitors has enabled us to identify a novel protein that is conserved over a wide range of eukaryotic organisms with tissue-specific expression related to specific functions of differentiated cells. The participation of this protein in signaling by the small GTPase Rac1 is an important although perhaps not unique aspect of its activity; B-ind1 and related proteins may have other cellular functions that remain to be discovered.

      Acknowledgments

      We thank Drs. G. Bokoch, A. Hall, and R. Davis for providing reagents. We also thank Dr. C. Gespach for support and the service center (INSERM SC6, Vesinet) for illustrations.

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