An Early Event in Adipogenesis, the Nuclear Selection of the CCAAT Enhancer-binding Protein β (C/EBPβ) mRNA by HuR and its Translocation to the Cytosol

HuR is a ligand for nuclear mRNAs containing adenylate-uridylate-rich elements in the 3′-untranslated region. Once bound to the mRNA, HuR is recognized by adapter proteins that then facilitate nuclear export of the complex. In the cytosol, HuR is thought to function to control stability and translation of its ligand message. In the 3T3-L1 cells HuR is constitutively expressed and localized predominantly to the nucleus in the preadipocytes. However, within 30 min of exposure to the differentiation stimulus the HuR content in the cytosol increases, consistent with HuR regulating the availability of relevant mRNAs for translation. Using in vitro RNA gel shifts, we have demonstrated that the CCAAT enhancer-binding protein β (C/EBPβ) message is a ligand for HuR. Within 2 h of initiation of the differentiation process, HuR complexes containing C/EBPβ mRNA could be isolated from the cytosolic compartment. Importantly, the process appears to be highly selective, as cyclin D1, which contains a putative HuR binding site and is expressed on the same time frame as C/EBPβ, was not found in the immunoprecipitated messenger ribonucleoprotein complexes. The proximity of this event to adipogenic stimuli and the importance of C/EBPβ to the differentiation process have led us to hypothesize a role for HuR in the regulation of the onset of adipogenesis. In support of this hypothesis, small interfering RNA suppression of HuR protein content resulted in an inhibition of C/EBPβ protein expression and an attenuation of the differentiation process. HuR is a ligand for nuclear mRNAs containing adenylate-uridylate-rich elements in the 3′-untranslated region. Once bound to the mRNA, HuR is recognized by adapter proteins that then facilitate nuclear export of the complex. In the cytosol, HuR is thought to function to control stability and translation of its ligand message. In the 3T3-L1 cells HuR is constitutively expressed and localized predominantly to the nucleus in the preadipocytes. However, within 30 min of exposure to the differentiation stimulus the HuR content in the cytosol increases, consistent with HuR regulating the availability of relevant mRNAs for translation. Using in vitro RNA gel shifts, we have demonstrated that the CCAAT enhancer-binding protein β (C/EBPβ) message is a ligand for HuR. Within 2 h of initiation of the differentiation process, HuR complexes containing C/EBPβ mRNA could be isolated from the cytosolic compartment. Importantly, the process appears to be highly selective, as cyclin D1, which contains a putative HuR binding site and is expressed on the same time frame as C/EBPβ, was not found in the immunoprecipitated messenger ribonucleoprotein complexes. The proximity of this event to adipogenic stimuli and the importance of C/EBPβ to the differentiation process have led us to hypothesize a role for HuR in the regulation of the onset of adipogenesis. In support of this hypothesis, small interfering RNA suppression of HuR protein content resulted in an inhibition of C/EBPβ protein expression and an attenuation of the differentiation process. When confluent 3T3-L1 preadipocytes are treated with differentiation inducers, they synchronously reenter the cell cycle and undergo approximately two rounds of cell division as they enter a process that has become known as mitotic clonal expansion (1Patel Y.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1279-1284Crossref PubMed Scopus (83) Google Scholar, 2Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (648) Google Scholar, 3Zhang J-W. Klemm D.J. Vinson C. Lane M.D. J. Biol. Chem. 2004; 279: 4471-4478Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This increase in cell number and commitment to differentiation mimics the increase in adipose mass in obesity (4Shepherd P.R. Gnudi L. Tozzo E. Yang H. Leach F. Kahn B.B. J. Biol. Chem. 1993; 268: 22243-22246Abstract Full Text PDF PubMed Google Scholar, 5Gnudi L. Shepherd P.R. Kahn B.B. Proc. Nutr. Soc. 1996; 55: 191-199Crossref PubMed Scopus (28) Google Scholar, 6Van R.L. Bayliss C.E. Roncari D.A. J. Clin. Investig. 1976; 58: 699-704Crossref PubMed Scopus (204) Google Scholar). In the 3T3-L1 preadipocytes, mitotic clonal expansion has been demonstrated to be required for terminal adipocyte differentiation (1Patel Y.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1279-1284Crossref PubMed Scopus (83) Google Scholar, 2Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (648) Google Scholar), and a transcription factor expressed prior to the start of the process, C/EBPβ, 1The abbreviations used are: C/EBPβ, CCAAT enhancer-binding protein; ARE, adenylate-uridylate-rich element; mRNP, messenger ribonucleoprotein; PPAR, peroxisome proliferator-activated receptor; Sc, Dharmacon siCONTROL non-targeting siRNA™ four-oligo pool; siRNA, small interfering RNA; UTR, untranslated region. plays an essential role in the mitotic clonal expansion as well as in subsequent events in the differentiation program (7Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 850-855Crossref PubMed Scopus (409) Google Scholar, 8Tang Q-Q. Lane M.D. Genes Dev. 1999; 13: 2231-2241Crossref PubMed Scopus (311) Google Scholar, 9Yeh W-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar). Transcription of the C/EBPβ gene is increased shortly after exposure of the cells to the differentiation inducers, and the C/EBPβ protein can be detected in the nucleus within 4 h (10Cole K.A. Harmon A.W. Harp J.B. Patel Y.M. Am. J. Physiol. 2004; 286: C349-C354Crossref PubMed Scopus (28) Google Scholar). On termination of the mitotic clonal expansion phase, C/EBPβ has been proposed to be responsible for the transactivation of C/EBPα and PPARγ genes, two transcription factors responsible for establishing and maintaining the adipocyte phenotype (11Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar, 12Hwang C. Loftus T. Mandrup S. Lane M. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (211) Google Scholar, 13Christy R.J. Kaestner K.H. Geiman D.E. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2593-2597Crossref PubMed Scopus (329) Google Scholar, 14Tang Q.Q. Jiang M.S. Lane M.D. Mol. Cell. Biol. 1999; 19: 4855-4865Crossref PubMed Scopus (128) Google Scholar, 15Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Rao M.S. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (606) Google Scholar, 16Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (193) Google Scholar). As C/EBPα expression is increased, C/EBPβ expression is attenuated (17Tang Q.Q. Jiang M.S. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13571-13575Crossref PubMed Scopus (36) Google Scholar). Thus, C/EBPβ plays a critical early regulatory role in the differentiation process. HuR is an RNA-binding protein belonging to the Hu/ELAV family of mRNA-binding proteins and is expressed in adipose (this article), intestine, spleen, thymus, and testis with minor expression in liver and uterus (18Lu J.-Y. Schneider R.J. J. Biol. Chem. 2004; 279: 12974-12979Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 19Ma W.J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. Chem. 1996; 271: 8144-8151Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). HuR contains a nucleocytoplasmic shuttling sequence and functions as an adapter protein in the nuclear export of mRNAs that contain adenylate-uridylate-rich elements (AREs) in their 3′-untranslated regions (19Ma W.J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. Chem. 1996; 271: 8144-8151Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 20Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (748) Google Scholar, 21Myer V.E. Fan X.C. Steitz J.A. EMBO J. 1997; 16: 2130-2139Crossref PubMed Scopus (282) Google Scholar, 22Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 24Peng S.S. Chen C.Y. Xu N. Shyu A.B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (656) Google Scholar). Depending on the specific message, the nuclear HuR-containing messenger ribonucleoprotein (mRNP) is bound by APRIL and/or pp32 and then CRM1, which is recognized by a specific binding domain on the nuclear pore complex, facilitating nucleocytoplasmic transport. An alternative to the CRM1 route is mediated by transportin-2 binding to the HuR mRNA complex for exit through the nuclear pore (25Brennan C.M. Steitz J.A. Cell. Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (888) Google Scholar, 26Gallouzi I.E. Steitz J.A. Science. 2001; 294: 1895-1901Crossref PubMed Scopus (235) Google Scholar). Once in the cytosol, HuR functions to stabilize mRNA ligands (27Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 28Jacobson A. Peltz S.W. Annu. Rev. Biochem. 1996; 65: 693-739Crossref PubMed Scopus (577) Google Scholar). Based on results obtained with the ectopic expression of the neuronal Hu family homolog HuB in both the 3T3-L1 adipocytes and human teratocarcinoma cells, HuR may also participate in control of translation initiation (29Jain R.G. Andrews L.G. McGowan K.M. Pekala P.H. Keene J.D. Mol. Cell. Biol. 1997; 17: 954-962Crossref PubMed Scopus (183) Google Scholar, 30Antic D. Lu N. Keene J.D. Genes Dev. 1999; 13: 449-461Crossref PubMed Scopus (179) Google Scholar). The selection of ligands and the translocation of HuR to the cytosol has been proposed to be under tight control (31Li H. Park S. Kilburn B. Jelinek M.A. Henschen-Edman A. Aswad D.W. Stallcup M.R. Laird-Offringa I.A. J. Biol. Chem. 2002; 277: 44623-44630Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Identification of the specific signal involved has remained elusive, with the exception of lipopolysaccharide-stimulated macrophages in which methylation of specific arginine residues in HuR by the nuclear coactivator-associated arginine methyltransferase (CARM) appears to control the process (31Li H. Park S. Kilburn B. Jelinek M.A. Henschen-Edman A. Aswad D.W. Stallcup M.R. Laird-Offringa I.A. J. Biol. Chem. 2002; 277: 44623-44630Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). HuR has been suggested to be involved in control of the cell cycle through stabilization of the mRNAs for cyclins A and B1 during S and G2, leading to increased expression of these proteins (32Wang W. Caldwell M.C. Lin S. Furneaux H. Gorospe M. EMBO J. 2000; 19: 2340-2350Crossref PubMed Scopus (396) Google Scholar). HuR involvement in differentiation was supported by RNA interference knockdowns of the protein in C2C12 cells, which resulted in an inhibition of myogenesis coincident with decreased expression of MyoD and myogenin mRNAs, known ligands for HuR (33van der Giessen K. Di-Marco D. Clair E. Gallouzi I.E. J. Biol. Chem. 2003; 278: 47119-47128Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Thus, it might be argued that HuR, by selecting specific mRNA ligands and controlling their expression, is capable of controlling critical events in the cell cycle as well as the differentiation process. In the current study we demonstrate that the C/EBPβ message is a ligand for HuR and that the time frame for cytosolic translocation of the complex is consistent with an early regulatory event in the differentiation process. Materials—Dulbecco's modified Eagle's medium was purchased from Invitrogen. Bovine calf serum and fetal calf serum were purchased from Hyclone Laboratories (Logan, Utah). The 3T3-L1 cells used in this work were obtained from Howard Green (Harvard University, Boston, MA). The BCA protein assay kit, the NE-PER™ cell fractionation kit, and the HALT™ protease inhibitor mix were from Pierce. The RNAqueous RNA extraction kit was from Ambion (Austin, TX). Reagents for molecular biology were purchased from Invitrogen. All other chemicals were of reagent grade and were purchased from Sigma-Aldrich. The 3A2 monoclonal antibody directed against HuR and the β-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) The mouse anti-Armenian and anti-Syrian hamster IgG1 monoclonal antibody used as an isotype control was obtained from directed against C/EBPβ were by M. Lane University, The antibody against was by Keene University, The reagent and the non-targeting were obtained from Dharmacon used for were the program All were to a to of and their were as C/EBPβ, and cyclin D1, and and and The binding site was in the used in the of for RNA gel by the sequence the of a 3T3-L1 Cell preadipocytes were and as (29Jain R.G. Andrews L.G. McGowan K.M. Pekala P.H. Keene J.D. Mol. Cell. Biol. 1997; 17: 954-962Crossref PubMed Scopus (183) Google Scholar). preadipocytes were 2 and adipocytes were used of was by exposure of the cells to and in the of fetal be to as the the of and NE-PER™ cell fractionation kit was used to cytosolic and nuclear from the 3T3-L1 cells as the with minor were used as a of for the In to the protease an inhibitor was to the time for the was increased from to Once the cytosolic was the nuclear was to a and the was to The isolated were of nuclear were prior to of complexes from nuclear cytosolic were immunoprecipitated and mRNA isolated and by a of the by Keene J.D. 2002; PubMed Scopus Google Scholar, in Scholar). were 4 in of and of cytosolic of nuclear was were for for the of RNA and as a with the protein in the were to an protease inhibitor complex inhibitor and The antibody was then for the isotype hamster IgG1 monoclonal antibody was for HuR, monoclonal 3A2 was the of was and the for h by the of protein with for an 2 h This was by two with The was then 4 and with in of the was this time for the and RNA was from the the Ambion RNAqueous kit as the RNA was was as J.M. Pekala P.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the in nuclear cytosolic HuR of nuclear protein and of cytosolic protein were used for RNA were from containing the the Ambion RNA and kit as the The was and used for gel of 2 of cytosolic nuclear protein was with the in the of binding and for were the antibody was this time and the binding was for an 30 min the of the binding was to a of and the was for a min An of gel and was to the The were for 30 min and then the was and complexes were on a gel in were under and to of the 3T3-L1 of the cells was Dharmacon reagent number a for HuR number by preadipocytes in were with as a to control were with and the other with Dharmacon siCONTROL non-targeting siRNA™ four-oligo number The cells were to the for which time the medium was with modified Eagle's medium with fetal after of the two of were and to an confluent essential for h after the the cells were to the differentiation were then for of the adipocyte HuR in the 3T3-L1 results in demonstrate the expression of HuR in and adipocytes as well as the of the NE-PER™ kit for cytosol nuclear this of nuclear and of cytosolic were and with antibodies directed against HuR, and The two were used as for nuclear and cytosolic be that the in β-tubulin with differentiation in has been D.A. M.A. Lane M.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In of protein were for the of both The demonstrate that HuR is expressed in the preadipocytes with expression as the cells is the increased of HuR in the cytosol with to of that in the preadipocytes of the HuR in the cytosol, in the adipocyte cytosolic HuR of HuR. that we are preadipocytes with adipocytes in protein is for The protein content of the during the differentiation process. then the time frame for HuR to the were and by for the of the HuR protein with to the time after exposure of the preadipocytes to the differentiation As in in the preadipocytes HuR is in the However, within 30 min after initiation of the cytosolic content increased by the h the cytosolic content of HuR a increase the of HuR was a and to the β-tubulin The increase was through the cells were to be not Thus, HuR the nucleus early and a the nucleus and the cytosol and that in the that the cytosolic the of which not during the time frame of the of the The C/EBPβ mRNA a for HuR in translocation of HuR to the cytosol after the of differentiation to the suggested that it might be involved in a critical early event in the differentiation process. on the role of HuR in the of specific mRNAs from the nucleus to the cytosol, we to for mRNA (10Cole K.A. Harmon A.W. Harp J.B. Patel Y.M. Am. J. Physiol. 2004; 286: C349-C354Crossref PubMed Scopus (28) Google demonstrated that the C/EBPβ protein can be found in the nucleus within 4 h of the of the differentiation to the 3T3-L1 preadipocytes. This that the C/EBPβ message exit the nucleus shortly after the of the differentiation of a HuR complex with the C/EBPβ message is for translocation to the cytosol, both HuR and C/EBPβ mRNA be to the cytosol after the of the of the C/EBPβ sequence the of an in the that might as a binding site for HuR. were from and the RNA gel a to the are in As in in the of protein the was to a molecular complex consistent with of a complex. of an HuR antibody resulted in a the of HuR in the complex. was antibodies against HuB were used in the In a of was used to for HuR Thus, in an in vitro assay HuR an complex with the from the of of and to the the C/EBPβ message was in an complex with HuR, were on both nuclear and cytosolic and 30 min after the of the differentiation As in that the HuR protein was detected in the nuclear both time However, the decreased nuclear of the HuR protein 30 min after of differentiation is of the mRNA in the immunoprecipitated complex in prior to the of the differentiation inducers, complexes could not be However, within 30 min of of the C/EBPβ mRNA was found to be in the immunoprecipitated complex. of the cytosol for the of these complexes the same time the of HuR protein in the cytosol with an increase 30 min after the of consistent with In the of the C/EBPβ mRNA in the immunoprecipitated complex the time is The in 4 support a in which of the preadipocytes with the differentiation results in of a message complex in the This is by translocation to the cytosol and a early event in the differentiation of the translocation time the of C/EBPβ mRNA in the complex 2 with a signal the time after a complex can be consistent with expression of the transcription factor not complex and are to in 4 with the control 4 adipocyte time and h after exposure to the differentiation transcription for the of are of with the of the selection of the C/EBPβ message by HuR, we for the of cyclin mRNA in the was it has a in the and is expressed during the differentiation program on the same time frame as C/EBPβ R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In we for the of mRNA, a message containing cyclin mRNA were detected not The demonstrate a highly HuR selection of the C/EBPβ mRNA as a early event in the differentiation process. of HuR on Dharmacon four-oligo we an to HuR protein content in the 3T3-L1 The study used two and a Dharmacon non-targeting four-oligo As under cells were with for which time the was with modified Eagle's medium fetal two were to confluent which was essential for of h after the the cells were to the differentiation demonstrated that HuR protein the time of with the differentiation were decreased by to the HuR content remained decreased for which time HuR protein to increase as the of the by The phenotype is and in The in A and the results of the cells for the of of both and were as demonstrated by of both and cells The decreased of the cells a and the of the in the However, the cells treated with HuR the this In treated to in A and was for HuR and C/EBPβ and The in demonstrate a suppression of HuR protein to both and results were with C/EBPβ the protein content of both the protein and protein of the transcription factor are decreased to of the expression of C/EBPα and as well as PPARγ that these transcription factors were also in the cells that the β-tubulin as a control in the is decreased to the by in results a the of HuR and C/EBPβ expression in and and the of the adipocyte in A and of these are consistent with the protein content of HuR controlling C/EBPβ expression the of the adipocyte results the expression of the RNA-binding protein HuR in the 3T3-L1 preadipocytes with a increase in protein content with to in the the of HuR protein is in the However, exposure of the cells to the differentiation is a of a nuclear complex by a translocation of the complex to the When HuR expression was siRNA, the cells their to of C/EBPβ, and not through a time are consistent with of HuR protein content and the of the cells to C/EBPβ and through the differentiation Importantly, work by has demonstrated that the differentiation process can be with the K.M. J.M. Biochem. J. 2002; PubMed Scopus (28) Google L. Biochem. Biophys. Res. Commun. 2001; 286: PubMed Scopus Google Scholar). However, in both differentiation was the C/EBPβ protein was expressed K.M. J.M. Biochem. J. 2002; PubMed Scopus (28) Google to L. Biochem. Biophys. Res. Commun. 2001; 286: PubMed Scopus Google Scholar). This that suppression of HuR expression results in an inhibition of differentiation a the of C/EBPβ are consistent with that the of the C/EBPβ message to the The proposed to the cytosolic translocation from the nucleus of the complex by adapter proteins in are recognized by specific X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (748) Google Scholar, W. H. T. M. S. Y. H. T. T. T. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, 26Gallouzi I.E. Steitz J.A. Science. 2001; 294: 1895-1901Crossref PubMed Scopus (235) Google Scholar, C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; PubMed Scopus Google Scholar). However, the C/EBPβ gene has and an alternative export which is mediated by HuR of the C.M. Gallouzi I.E. Steitz J.A. J. Cell Biol. 2000; PubMed Scopus Google Scholar). This selection of the C/EBPβ mRNA by HuR and of its translocation to the cytosol a critical control in the onset of adipogenesis. In these are consistent with other cytosolic translocation of Hu proteins and their mRNAs with the onset of myogenesis (33van der Giessen K. Di-Marco D. Clair E. Gallouzi I.E. J. Biol. Chem. 2003; 278: 47119-47128Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Keene J.D. 2002; PubMed Scopus Google Scholar). work from demonstrated that ectopic of neuronal HuB in the 3T3-L1 preadipocytes resulted in an early expression of C/EBPβ by a onset of differentiation and increased of (29Jain R.G. Andrews L.G. McGowan K.M. Pekala P.H. Keene J.D. Mol. Cell. Biol. 1997; 17: 954-962Crossref PubMed Scopus (183) Google Scholar, R.G. Pekala P.H. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). suggested that an of the Hu family may a role in the control of the differentiation process. The that HuR that The of a ligand mRNA by HuR is a process. cyclin D1, an mRNA expressed with the same as C/EBPβ R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), a message an ARE, was in the complexes. (32Wang W. Caldwell M.C. Lin S. Furneaux H. Gorospe M. EMBO J. 2000; 19: 2340-2350Crossref PubMed Scopus (396) Google have that HuR in from cells not to the cyclin in gel This was the as have demonstrated that HuR to the cyclin in gel that the of the cell may control the selection of ligands by the HuR that the tight control of specific mRNA selection and translocation to the cytosol during the differentiation process be an and that these support the of a control in the differentiation process. a of HuR protein is with as as of the in the cytosol, after the expression of C/EBPβ has work has in the the mRNA is a ligand for HuR and that other ligand mRNAs are with to the time of differentiation and P. H. in of the on RNA and and Scholar). U. and P. H. in that HuR not functions in establishing the adipocyte phenotype in its and Keene for the that in the of