B-cell Receptor Activation Induces BIC/miR-155 Expression through a Conserved AP-1 Element

microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site ∼40 bp upstream from the site of initiation but not an upstream NF-κB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155. microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site ∼40 bp upstream from the site of initiation but not an upstream NF-κB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155. MicroRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNABICB-cell integration clusterBCRB-cell receptorTLRToll-like receptorERKextracellular signaling-regulated kinaseJNKc-Jun NH2-terminal kinaseMEKmitogen-activated protein kinase/ERK kinaseTNFtumor necrosis factorRTreverse transcriptionqRTquantitative RTG3PDHglyceraldehyde-3-phosphate dehydrogenaseRACErapid amplification of cDNA endsRIPAradioimmune precipitation bufferEBVEpstein-Barr virusESTexpressed sequence tagILinterleukin. have been shown to be key mediators of cell regulatory processes such as those controlling cell growth, differentiation, and development (1Hwang H.W. Mendell J.T. Br. J. Cancer. 2006; 94: 776-780Crossref PubMed Scopus (989) Google Scholar, 2Miska E.A. Curr. Opin. Genet. Dev. 2005; 15: 563-568Crossref PubMed Scopus (726) Google Scholar, 3Wienholds E. Plasterk R.H. FEBS Lett. 2005; 579: 5911-5922Crossref PubMed Scopus (678) Google Scholar). The ability of at least some miRNAs to significantly alter cell processes and cell fate is attributable to their capacity to influence the expression of a large number of target mRNA species. miRNAs function to inhibit translation of mRNAs through specific but imperfect base pairing with their 3′-untranslated regions. The binding of miRNA protein complexes to mRNAs results in localization of the miRNA-protein-mRNA complex to a perinuclear compartment referred to as GW or P bodies, thereby preventing access to ribosomes, and in some cases, leading to the degradation of the respective mRNA (4Liu J. Rivas F.V. Wohlschlegel J. Yates J.R. II I Parker R. Hannon G.J. Nat. Cell Biol. 2005; 7: 1261-1266Crossref PubMed Scopus (514) Google Scholar, 5Liu J. Valencia-Sanchez M.A. Hannon G.J. Parker R. Nat. Cell Biol. 2005; 7: 719-723Crossref PubMed Scopus (969) Google Scholar). microRNA B-cell integration cluster B-cell receptor Toll-like receptor extracellular signaling-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein kinase/ERK kinase tumor necrosis factor reverse transcription quantitative RT glyceraldehyde-3-phosphate dehydrogenase rapid amplification of cDNA ends radioimmune precipitation buffer Epstein-Barr virus expressed sequence tag interleukin. The microRNA, miR-155, is processed from a primary transcript, referred to as B-cell integration cluster (BIC), whose upstream region was originally identified as a common site of integration of the avian leukosis virus in lymphomas (6Clurman B.E. Hayward W.S. Mol. Cell. Biol. 1989; 9: 2657-2664Crossref PubMed Scopus (153) Google Scholar). Transgenic mouse studies demonstrated that B-cell targeted expression of BIC leads to the development of B-cell malignancies (7Costinean S. Zanesi N. Pekarsky Y. Tili E. Volinia S. Heerema N. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7024-7029Crossref PubMed Scopus (960) Google Scholar). Further, a number of miRNA profiling studies have shown elevation of miR-155 in a wide array of cancers including lymphomas (7Costinean S. Zanesi N. Pekarsky Y. Tili E. Volinia S. Heerema N. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7024-7029Crossref PubMed Scopus (960) Google Scholar, 8Kluiver J. Poppema S. de Jong D. Blokzijl T. Harms G. Jacobs S. Kroesen B.J. van den Berg A. J. Pathol. 2005; 207: 243-249Crossref PubMed Scopus (583) Google Scholar, 9van den Berg A. Kroesen B.J. Kooistra K. de Jong D. Briggs J. Blokzijl T. Jacobs S. Kluiver J. Diepstra A. Maggio E. Poppema S. Genes Chromosomes Cancer. 2003; 37: 20-28Crossref PubMed Scopus (218) Google Scholar, 10Volinia S. Calin G.A. Liu C.G. Ambs S. Cimmino A. Petrocca F. Visone R. Iorio M. Roldo C. Ferracin M. Prueitt R.L. Yanaihara N. Lanza G. Scarpa A. Vecchione A. Negrini M. Harris C.C. Croce C.M. 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To date, miR-155 is one of the most highly implicated microRNAs in cancers. miR-155 has been shown to play a critical role in lymphocyte activation in vivo (15Thai T.H. Calado D.P. Casola S. Ansel K.M. Xiao C. Xue Y. Murphy A. Frendewey D. Valenzuela D. Kutok J.L. Schmidt-Supprian M. Rajewsky N. Yancopoulos G. Rao A. Rajewsky K. Science. 2007; 316: 604-608Crossref PubMed Scopus (1279) Google Scholar, 16Rodriguez A. Vigorito E. Clare S. Warren M.V. Couttet P. Soond D.R. van Dongen S. Grocock R.J. Das P.P. Miska E.A. Vetrie D. Okkenhaug K. Enright A.J. Dougan G. Turner M. Bradley A. Science. 2007; 316: 608-611Crossref PubMed Scopus (1613) Google Scholar) and is induced by a number of immune cell stimuli including Toll-like receptor (TLR) ligands, tumor necrosis factor-α (TNF-α), interferon-β, and antigen (B-cell receptor (BCR) engagement) (9van den Berg A. Kroesen B.J. Kooistra K. de Jong D. Briggs J. Blokzijl T. Jacobs S. Kluiver J. Diepstra A. Maggio E. Poppema S. Genes Chromosomes Cancer. 2003; 37: 20-28Crossref PubMed Scopus (218) Google Scholar, 17O'Connell R.M. Taganov K.D. Boldin M.P. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1604-1609Crossref PubMed Scopus (1568) Google Scholar, 18Taganov K.D. Boldin M.P. Chang K.J. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12481-12486Crossref PubMed Scopus (3569) Google Scholar). The mechanisms through which miR-155 is regulated following TLR and interferon signaling in macrophages has recently come under study (17O'Connell R.M. Taganov K.D. Boldin M.P. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1604-1609Crossref PubMed Scopus (1568) Google Scholar). In this study, TLR ligand-mediated activation of miR-155 was shown to occur through myeloid differentiation factor 88 (MyD88) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-dependent pathways (17O'Connell R.M. Taganov K.D. Boldin M.P. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1604-1609Crossref PubMed Scopus (1568) Google Scholar). Interferon signaling was found to require an autocrine pathway involving TNF-α. Lastly, induction of miR-155 by poly(I-C) and TNF-α were shown to be inhibited by a Jun-activated kinase (JNK) inhibitor, suggesting that this pathway plays a role in induction of miR-155 in these systems. Here we have begun to analyze signaling pathways involved in activating miR-155 following B-cell receptor (BCR) engagement. This analysis identified critical pathways required for BCR-mediated miR-155 activation, some of which likely overlap with pathways activated by TLR and TNF-α signaling in macrophages. Together these studies define some of the fundamental miR-155 regulatory processes following immune cell activation and lays the groundwork for understanding some of the mechanisms through which BIC/miR-155 is overexpressed in tumors. Cell Culture and Treatments—The EBV-negative human Burkitt lymphoma cell line, Ramos, was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (Invitrogen). B-cell receptor cross-linking experiments were carried out by exposure to anti-human-IgM (Sigma, catalog number I 0759). An equal volume of fresh medium was added to Ramos cells (which were at densities of ∼1–2 × 106/ml) 1 day before treatment. On the day of treatment, cells were counted, and 2 × 107 cells were added to 10 ml of fresh complete RPMI medium containing 10 μm anti-IgM. Inhibitor experiments were carried out as above except that the inhibitors (all purchased from Calbiochem®) were added to cells resuspended in fresh medium 30 min prior to the addition of anti-IgM (to a final concentration of 10 μm). For RT-PCR analysis, cells were harvested 24 h following the addition of anti-IgM. For Western blot analysis, cells were treated as above except that cells were harvested at the indicated the for and of JunB, c-Fos, and FosB is inhibited by ERK and JNK inhibitors. Ramos cells were with the indicated inhibitors prior to the addition of anti-IgM. were and to Western blot analysis as under JNK JNK p38 p38 and was a number to the For primary BIC analysis, 2 of was to cDNA catalog number were carried out the following and BIC BIC reverse P.S. Tam W. Sun L. Chadburn A. Li Z. Gomez M.F. Lund E. Dahlberg J.E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3627-3632Crossref PubMed Scopus (1206) Google and G.J. J. Virol. PubMed Scopus Google Scholar). were the following for min by of 30 and was with 2 of cDNA catalog number and an miR-155 expression was a miRNA catalog number with the miR-155 catalog number to the was carried out the following for min by of and 30 The expression of BIC and miR-155 in was by the in which is the and of miR-155 of sequence upstream and of sequence from the miR-155 site in were used to the mouse the for with shown in was identified as the and to and of the mouse was carried out from the human B-cell line, Ramos, following exposure to a cDNA amplification to the The which is to the of the human BIC/miR-155 primary was with a of bp was and the by were to the most in The site of the human BIC was found to be located at of the human and of BIC/miR-155 human BIC/miR-155 promoter from to to the site was from by the and The was with and and and The promoter region was and were identified to the of the miR-155 was carried out a II the following and and and and In the transcription factor binding site was with an were by with and by sequence Reporter × Ramos cells were of containing 10 ml of RPMI 1640 fetal bovine of the respective of the were with ml of ml of of ml of was added to of and the were and at for For ml of was added to of containing and were at for 24 this human number was added to a final concentration of 10 μm to the for were harvested 24 h and for are as the of The shown in were carried out as above except that cells were not treated with anti-IgM and cells were with 1 of a JunB, or FosB expression or of JunB of FosB expression The of anti-IgM treated cells J. C. M. J. Z. E. J. Virol. 2007; Scholar) were carried out as for the anti-IgM analysis in Ramos cells except that cells were by Western cells were treated as above prior to for Western blot For cell cells were in buffer and for 10 min at to To × 107 Ramos cells were in of buffer 10 1 and of 10% was and the was for 10 The were to a and for 2 min The was and of buffer 1 1 and and was and the was by The were a for min at and for min in a The was to and for in Western blot number ERK number c-Jun number and catalog number number and FosB catalog number JunB and number were used for Western blot Western was carried out of cell of cell was carried out an 2 × 107 cells were in of buffer 2 and for 1 h at were at for 10 min at and the were to Cell were with of protein or protein for 1 the of the FosB catalog number and of the JunB catalog number were with of protein or in of buffer for 1 h at and with The were and the were to the complexes and a at The were with buffer with for of buffer was added to and were for min at and an for Western blot cells were cultured for 2 h in the or of 10 μm anti-IgM (Sigma, catalog number I × 107 cells were used for were with for 30 was added to a final concentration of and cells were were with Cell were in buffer and for 1 Cell were to were in a at for and was used for For of was with of protein with a at were and was to containing of protein for a were and was to a containing the following FosB number JunB number of number and of number were at and of protein was added for a at were and was were with were with of containing at for 1 of 10% 2 of 1 were and were for h at with were at and resuspended in of 1 was used for the following for 30 for 30 for 30 for 30 used were as BIC promoter and and BIC 1 and binding 10 of was with of binding number AP-1 were purchased from AP-1 and AP-1 were purchased from and AP-1 experiments were carried out the following JunB FosB and and experiments were by with in binding buffer for 10 which was added for a at were a and for h at were an of BIC/miR-155 following BCR the ERK and JNK identify pathways involved in BIC promoter activation following BCR activation, the human B-cell line, Ramos, was treated with anti-IgM in the of or p38 pathway inhibitors. were treated with or inhibitors of the upstream ERK mitogen-activated protein kinase kinase or the JNK inhibitor, or the p38 inhibitor, for h prior to the addition of anti-IgM. 24 h cells were was and the was to RT-PCR to the of BIC and miR-155 shown in the inhibitors and the JNK inhibitor, suppressed induction of BIC as as miR-155 the p38 inhibitor, has These that activation of BIC transcription is through the ERK and JNK of the BIC in Ramos expression is highly regulated at the transcription cDNA BIC has been identified as a with the miR-155 sequence located in W. D. Hayward W.S. Mol. Cell. Biol. PubMed Google Scholar). an analysis of the base identified an that an located bp upstream from the most the for the and of this with This that BIC be of that be promoter in carried out from Ramos cells following exposure to anti-IgM. were and of these the upstream This analysis that at least in activated the BIC is of with the initiation site located at of with most of the identified as as the cDNA W. D. Hayward W.S. Mol. Cell. Biol. PubMed Google Scholar). Further, a sequence is located 24 bp upstream from the site which is the that these in of BIC conserved regulatory in the BIC a the mouse base was carried out the human BIC site bp upstream to bp was identified that is the the are located mouse upstream from the mouse This is to the bp the human miR-155 sequence and the human indicated in the promoter The human BIC promoter was for transcription factor binding the T. E. I. E.A. Res. PubMed Scopus Google Scholar). This analysis identified an AP-1 site and a c-Ets site with the region is a NF-κB site located bp upstream from the human BIC transcription site the AP-1 and c-Ets are conserved in the NF-κB site is not is a putative NF-κB site located to the region upstream from the c-Ets site The AP-1 for of BIC following BCR putative and c-Ets were to be for or to the activation of BIC following activation of the To the of these in BIC the human BIC promoter was upstream from a and were at of these promoter or were Ramos cells or an EBV-negative of the cell line, J. C. M. J. Z. E. J. Virol. 2007; and were or with an anti-IgM shown in of the NF-κB site influence promoter activation, and of the c-Ets site a influence promoter In of the AP-1 site promoter and response to BCR activation. These results that the AP-1 site plays a role in BIC promoter following activation of B-cell receptor of the AP-1 c-Fos, and FosB, the ERK and JNK of the AP-1 of transcription that be for induction of the BIC promoter through the AP-1 site by JunB, c-Fos, and FosB expression by Western blot Using cell or we were to c-Jun that c-Jun and or the or In were at 2 h induction of and FosB were at 2 h and through h The expression of c-Fos, and FosB were in the of and p38 inhibitors. shown in induction is significantly inhibited by the inhibitor, and inhibited by the inhibitor, and the JNK inhibitor, FosB expression was in the of of these inhibitors. The of JunB was by the and JNK the was In the p38 inhibitor, influence the expression of of these AP-1 These results show that the and JNK pathways induced expression of the AP-1 JunB, c-Fos, FosB, suggesting that these play a role in the induction of BIC JunB and FosB to the BIC following B-cell experiments demonstrated and FosB or that these likely occurs following BCR activation in this The binding of JunB and FosB to the BIC promoter following B-cell receptor was by chromatin immunoprecipitation that or the of were the of was not by BCR activation, of the were from cells to anti-IgM This in the chromatin immunoprecipitation and that the chromatin at the BIC promoter is activated following BCR activation. Using the and precipitation of the BIC promoter was that JunB and FosB to the BIC promoter following activation of the B-cell In was in FosB or JunB that a located in from the site of transcription initiation FosB and JunB to the BIC promoter following BCR activation and likely play a role in the activation of BIC To that JunB and FosB to the AP-1 site of the BIC analysis was carried out a the AP-1 An is from Ramos cells treated with that is not in from Ramos cells This is by but not a containing the AP-1 site used in the shown in of this is a JunB and this is an FosB These that this induced is of JunB, which is likely with the of a complete of this with the FosB is to binding of the or to a of this JunB to AP-1 such as is not at this these that JunB is a of this complex and that FosB is a Lastly, experiments a BIC or the AP-1 with JunB, FosB, or JunB FosB expression were carried out to that the binding of these to the AP-1 site is of promoter activation. of JunB or FosB expression the of the JunB and FosB expression results in a induction induction was the AP-1 site that activation likely occurs through the binding to the conserved AP-1 The analysis identified the and a for BIC in a human B-cell the of the of the to some that this a was from a of and the that be promoter in suggesting added to BIC of the promoter in the B-cell line, Ramos, is not specific to this cell analysis of B-cell line that of BIC to the of the and E. is likely that this promoter is used in and shown in of the ERK or the JNK pathways the of and FosB This likely occurs through and of the factor which is a critical of and FosB promoter activation the binding to the of P.S. K. T. R. Res. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, R. R. J. J. PubMed Scopus Google Scholar). In is for of FosB A. J. Biol. Full Text Full Text PDF PubMed Scopus Google which to the activation of FosB of the ERK or JNK pathways an the of the JunB suggesting a role in a of is that in addition to the of FosB, this in the of JunB to the of AP-1 by ERK and JNK inhibitors. these results for a key role in and activation of FosB and JunB in the induction of BIC expression following BCR activation. number of studies have demonstrated expression of BIC in cells the of J. Genes Chromosomes Cancer. 2006; PubMed Scopus Google Scholar, J. E. de Jong D. Blokzijl T. Jacobs S. Kroesen B.J. Poppema S. van den Berg A. Genes Chromosomes Cancer. 2006; PubMed Scopus Google Scholar). This not that are for the induction of JunB is a established target of that is induced by K. A. E. J. Virol. PubMed Scopus Google Scholar). is that BIC expression at least in through the induction of The shown in that the AP-1 site is for transcription and activated that is a in BIC we a response to BCR activation with the AP-1 site suggesting that promoter with AP-1 to BIC This is to be to of the AP-1 site the of this were but in response was the c-Ets site located at the site was suggesting the that this site to activation, through the binding of the In is a site R. R. J. J. PubMed Scopus Google Scholar) located at to which play a role in activation in response to BCR and TLR activation. that the AP-1 site is an of promoter are likely to to promoter function and play a role in BIC promoter under these or activating (17O'Connell R.M. Taganov K.D. Boldin M.P. Cheng G. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1604-1609Crossref PubMed Scopus (1568) Google Scholar) have shown that the JNK pathway is required for activation of BIC expression following the activation of TLR signaling the of ERK or the FosB, c-Fos, or JunB AP-1 is not is likely that the BIC AP-1 site is for induction of BIC this has not to been studies the overlap is these pathways in the activation of BIC/miR-155 AP-1 signaling plays a key role in in the immune and is for the induction of regulatory In AP-1 activation plays a role in immune cell activation and For Jacobs D. I. A. PubMed Scopus Google Scholar) that the FosB under the of a promoter in BIC/miR-155 a in immune cell activation (15Thai T.H. Calado D.P. Casola S. Ansel K.M. Xiao C. Xue Y. Murphy A. Frendewey D. Valenzuela D. Kutok J.L. Schmidt-Supprian M. Rajewsky N. Yancopoulos G. Rao A. Rajewsky K. Science. 2007; 316: 604-608Crossref PubMed Scopus (1279) Google Scholar, 16Rodriguez A. Vigorito E. Clare S. Warren M.V. Couttet P. Soond D.R. van Dongen S. Grocock R.J. Das P.P. Miska E.A. Vetrie D. Okkenhaug K. Enright A.J. Dougan G. Turner M. Bradley A. Science. 2007; 316: 608-611Crossref PubMed Scopus (1613) Google Scholar). is likely that the induction of BIC/miR-155 plays a key role in at least some of the of AP-1 activation. the AP-1 and is and AP-1 tumor growth, and through a wide array of The highly implicated of miR-155 in that induction of BIC/miR-155 be a critical of AP-1 signaling that to AP-1 oncogenic for the of this for the and and for immunoprecipitation and chromatin immunoprecipitation