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EBP-β expression in 3T3-L1 adipocytes
BBRC Biochemical and Biophysical Research Communications 313 (2004) 619–622 www.elsevier.com/locate/ybbrc
HuB localizes to polysomes and alters C/EBP-b expression in 3T3-L1 adipocytes Kira R. Gantt, Renu G. Jain, Ronald W. Dudek, and Phillip H. Pekala* Department of Biochemistry and Molecular Biology, The Brody School of Medicine, Greenville, NC 27858, USA Received 26 November 2003
Abstract The RNA binding protein HuB was ectopically expressed in 3T3-L1 preadipocytes and localized primarily to the nucleus, as the cells differentiated HuB redistributed to the cytosol and on analysis localized to the dense polysomes. Electron micrographs confirm association of HuB with the ribosomes in the adipocytes consistent with a proposed role in control of translation and mRNA stability. Examination of the expression of C/EBP-b in the cells expressing HuB relative to the parental 3T3-L1 adipocytes demonstrated an alteration in the LAP to LIP ratio. The data support a role for endogenous Hu proteins in the differentiation process, potentially affecting the rate of differentiation by controlling the concentration of the dominant negative transcription inhibitor, LIP. Ó 2003 Elsevier Inc. All rights reserved. Keywords: 3T3-L1; Adipocytes; C/EBP-b; Differentiation; HuB; mRNA stability; Translation
HuB is an mRNA binding protein belonging to the RNA recognition motif family [1]. HuB binds to A + U rich elements (AREs) in the 30 UTR of various mRNAs controlling stability and translatability of the message [2,3]. In vitro RNA gel shifts demonstrated that an ARE in the 30 UTR of the GLUT1 mRNA was a ligand for HuB and ectopic expression of HuB in the 3T3-L1 cells led to an acceleration of the differentiation process and a 10-fold overexpression of the GLUT1 glucose transporter protein [2]. Interestingly, while both HuB and GLUT1 were expressed in the preadipocytes, no alteration of transporter expression occurred. This led us to propose that HuB remained sequestered in the nucleus prior to the initiation of differentiation whereupon it chaperoned mRNAs that may play a role in establishing and maintaining the adipocyte phenotype to the cytosol and controlled their expression. In the current study, we demonstrate that in the adipocytes, ectopically expressed HuB localizes to the ribosomes and alters expression of C/EBP-b relative to the parental 3T3-L1 cells. * Corresponding author. Fax: 1-252-744-3383. E-mail address: [email protected] (P.H. Pekala).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.162
Materials and methods Materials. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco/Invitrogen (Grand Island, New York). 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). All other chemicals were of reagent grade and purchased from Sigma– Aldrich Biochemical (St. Louis, MO). Antibody against the G10 tag (T7) was purchased from Novagen (Madison, WI). An isotype control IgG2 a was obtained from BD-Pharmingen (San Diego, CA). AuroProbe anti-mouse antibody labeled with 15 nm gold particles was purchased from Amersham (Piscataway, NJ). Antibodies directed against C/EBP-b and HuB were provided by M. Daniel Lane (The Johns Hopkins University, Baltimore, MD) and Jack Keene (Duke University, Durham, NC), respectively. 3T3-L1 cell culture and transfection. 3T3-L1 preadipocytes were cultured, maintained, and differentiated as previously described [2]. For experimentation preadipocytes were harvested at 2 days postconfluence and adipocytes were used at eight days postinduction of differentiation. Cells were transfected with pBC-HuB expression vector, selected for, and characterized as previously described [2]. The HuB cDNA contained a gene 10 (g10) epitope tag for immunoidentification. Polysome profiles: analysis of polysomes by sucrose density gradients. Polysome profiles were generated as described by Long and Pekala [4]. Western blot analysis. Western blot analysis was performed as previously detailed [5].
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Electron microscopy. HuB transfectants (preadipocytes or adipocytes) were collected by scraping, isolated by centrifugation (1600 rpm for 5 min), and fixed (4% formaldehyde, 0.1% glutaraldehyde in phosphate buffered saline, pH 7.4). The fixed cells were dehydrated by 10 min treatments with graded concentrations of ethanol (70%, 90%, 95%, and 100%), embedded in LR White plastic, and ultrathin sections were cut with a Reichter ultramicrotome (Reichter Otische Werke, Vienna, Austria). Immuno-cytochemistry was performed on sections using the monoclonal antibody to the T7 gene 10 epitope tag or an isotype control IgG2 a at 1:100 dilutions in the blotting solution. Secondary anti-mouse antibody conjugated with 15 nm gold particles was used for visualization. Blocking was accomplished with egg albumin. Sections were incubated in 2% OsO4 in 0.2 M phosphate buffer for 1 h and washed with phosphate buffered saline and then with distilled water. Sections were counterstained with 8% uranyl acetate in 50% ethanol and visualized with the JEOL 1200EX electron microscope operated at 60 kV. HuB nomenclature. HuB is the mammalian homolog of the Drosophilia embryonic lethal abnormal vision gene product and has been referred to in the previous literature as human embryonic lethal abnormal vision like neuronal protein 1 (Hel-N1). For the sake of consistency with contemporary literature we will refer to the gene and protein product as HuB.
Results and discussion Our previous studies demonstrated that ectopic expression of HuB altered the distribution of GLUT1 mRNA among ribosomes and polysomes, shifting the bulk of the message to the most dense fractions of the gradient. Remarkably, initiation complexes could not be detected, suggesting that HuB expression resulted in an acceleration of the initiation process and the loading of more ribosomes per polysome [2]. In the current study sucrose density gradient analysis was performed to determine if the ectopically expressed HuB localized to the same fractions within the 3T3-L1 adipocytes. The analysis was performed on day 8 following induction of differentiation and a representative polysome profile is presented in Fig. 1. In Fig. 1A, the profile depicts fractionation of the protein synthesis apparatus. Western blot analysis performed on protein isolated from selected fractions shown in Fig. 1B indicates that the bulk of the HuB protein can be found in the very dense fraction of the gradient. The distribution appears identical to that we have previously published for GLUT1 message in HuB expressing cells [2]. To further investigate the association of HuB protein with the ribosomes, electron microscopy was performed. As demonstrated in Fig. 2A, no immunogold staining was observed to be associated with the ribosomes in the preadipocytes. However, Fig. 2B, presenting samples derived from adipocytes, clearly demonstrates the colocalization of the immunogold and the ribosomes (see arrow). Thus, in the adipocytes, HuB co-sediments with dense polysomes in a sucrose gradient and can be visualized by electron microscopy as localized to ribosomal
Fig. 1. Polysome profile and HuB localization in 3T3-L1 adipocytes. (A) Polysome profile from HuB transfected cells. A postmitochondrial supernatant was prepared from HuB transfected cells on day 8 of a differentiation time course and applied to a 15–40% sucrose density gradient, which was centrifuged at 20,000 g for 2 h. The gradients were fractionated and the absorbance at 250 nm was measured generating a polysome profile. The top of the gradient is on the left. (B) HuB localization in polysome profile. Protein was extracted from the fractions and Western blot analysis was performed for HuB. Fraction numbers in the Western blot (B) correspond to fraction numbers in the polysome profile (A). The data presented are representative of duplicate determinations performed on each of two separate transfections.
structures, supporting a relationship between HuB and the translation apparatus. While our previous studies have focused on the effect of HuB on GLUT1 expression, we do not believe that the GLUT1 message is a ligand for HuB in isolation. Our model would suggest that HuB selects multiple mRNAs or perhaps families of related messages essential for establishing and maintaining the adipose phenotype, chaperones them to the cytosol, and controls their expression. Examination of differentiation related genes that might prove to be ligands for HuB yielded C/EBP-b. This transcription factor is expressed early in the differentiation program, with protein detected in the nucleus within 4 h of exposure of the cells to the differentiation stimuli [6]. Examination of the message demonstrates the presence of a strong ARE between bases 1291 and 1400 of the 30 UTR. If HuB did bind the C/EBP-b message, we would predict that much like GLUT1 an alteration in expression should be observed. Examination of C/EBP-b expression by Western blot analysis in the parental versus the HuB transfectants (Fig. 3) demonstrates that the HuB expressing cells display an altered expression of this transcription factor. Most notably is the alteration of the expression of the truncated form of C/EBP-b, LIP (Fig. 3B). As opposed
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Fig. 2. Localization of HuB by electron microscopy. Electron micrographs of HuB staining in preadipocyte and adipocyte cytosol. (A) Preadipocyte cytosol shows the presence of ribosomes (dark grey, triangular-shaped structures) arranged in coils, but no staining for HuB. Ribosomes are magnified in inset A. (B) Adipocyte cytosol shows the presence of HuB visualized with 15 nm gold particles closely associated with ribosomes. Inset B shows gold particles (black, rounded-shaped structures) superimposed on coiled ribosomes. Osmium/uranyl acetate staining was done to visualize cytosolic structures. The T7 gene 10 antibody was used to immuno-stain for HuB, with a goat anti-mouse 15 nm gold conjugate secondary. Use of an isotype control antibody showed no staining. Magnification: 25,000.
Fig. 3. Effect of HuB expression on C/EBP-b expression. (A) Cell protein extracts equivalent to 106 cells were prepared from parental 3T3-L1 cells during a differentiation time course and subjected to immunoblot analysis for C/EBP-b protein content as described in Materials and methods. The expression patterns for the full length form of the transcription factor C/EBP-b, also known as LAP (32 kDa) and the truncated dominant negative inhibitory form of C/EBP-b also known as LIP (21 kDa) are shown. (B) C/EBP-b expression in the HuB transfectants. The data are representative of duplicate determinations performed on each of two separate transfections.
Fig. 4. Effect of HuB expression on C/EBP-a expression. (A) Cell protein extracts equivalent to 106 cells were prepared from parental 3T3-L1 cells during a differentiation time course and subjected to immunoblot analysis for C/EBP-a protein content as described in Materials and methods. The expression patterns for the full length form of the transcription factor C/EBP-a, also known as 42 kDa and the truncated form 30 kDa are shown. (B) C/EBP-a expression in the HuB transfectants. The data are representative of duplicate determinations performed on each of two separate transfections.
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to the parental 3T3-L1 cells, LIP is expressed over a much shorter duration, detectable at day 0, maximum at day 4 with expression suppressed by day 7. This provides a significantly increased LAP (full length form) to LIP ratio in the HuB transfectants. This would suggest that HuB controls the LAP to LIP ratio through binding to the ARE and influencing translation. As LIP functions as a dominant negative C/EBP family member one might predict that due to the shorter duration of LIP expression and the LAP to LIP ratio, the differentiation process might be accelerated/enhanced in the HuB expressing cells. Indeed, examination of the expression of C/EBP-a (Fig. 4) confirms this prediction. In the 3T3-L1 parental cells (Fig. 4A), C/EBP-a expression is detected at day 5 with significant accumulation occurring between days 7 and 8. While in the HuB transfected cells (Fig. 4B), C/EBP-a expression can be detected at day 4 with significant accumulation by day 5. As C/EBP-b, at least in part, is thought to control expression of the C/ EBP-a gene, the earlier expression of the b-isoform and the increased LAP to LIP ratio in the HuB transfectants (Fig. 3) would be consistent with an earlier and robust expression of C/EBP-a. Finally, we note that examination of C/EBP-d expression indicated no differences between parental cells and the HuB transfectants (R. Jain and P.H. Pekala, unpublished results). We initiated experiments ectopically expressing HuB, a neuronal protein, in the 3T3-L1 cells [2] as previous attempts to transfect various mammalian cell lines with HuB resulted in cessation of cellular proliferation and/or dramatic morphological changes precluding analysis of specific effects of HuB. Those studies did indeed provided insight into the mechanism by which HuB controlled expression of its ligand mRNAs. We have since described the endogenous expression of another member of the Hu family in the adipocytes, HuR [7] and demonstrated using in vitro RNA gel shifts that the ARE contained in the 30 UTR of the C/EBP-b mRNA is a ligand for HuR (R. Tenney and P.H. Pekala, unpublished
results). Taken together these observations begin to provide insight into the function of HuR in adipocyte differentiation.
Acknowledgments The authors thank Jack Keene for thoughtful discussions of the work and acknowledge the expert technical assistance of Dan Whitehead in the performance of the electron microscopy and the grant support of the NIH (DK55769) and the American Diabetes Association.
References [1] T.D. Levine, F. Gao, P.H. King, L.G. Andrews, J.D. Keene, HelN1: an autoimmune RNA-binding protein with specificity for 30 urydilate-rich untranslated regions of growth factor mRNAs, Mol. Cell. Biol. 13 (1993) 3494–3504. [2] R.G. Jain, L.G. Andrews, McGowan, P.H. Pekala, J.D. Keene, Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes, Mol. Cell. Biol. 17 (1997) 954–962. [3] D. Antic, N. Lu, J.D. Keene, ELAV tumor antigen, Hel-N1 increases translation of neurofilament M mRNA and induces formation of neuritis in human teratocarcinoma cells, Genes Dev. 13 (1999) 449–461. [4] S. Long, P.H. Pekala, Regulation of GLUT4 mRNA stability by TNF-a: alterations in both protein binding to the 30 untranslated region and initiation of translation, Biochem. Biophys. Res. Commun. 220 (1996) 949–953. [5] J.M. Stephens, P.H. Pekala, Transcriptional repression of the C/EBPa and GLUT4 genes in the 3T3-L1adipocytes by tumor necrosis factor-a. Regulation is coordinate and independent of protein synthesis, J. Biol. Chem. 267 (1992) 13580–13584. [6] K.A. Cole, A.W. Harmon, J. Harp, Y.M. Patel, Rb regulates C/EBPb-DNA-binding activity during 3T3-L1 adipogenesis, Am. J. Cell. Physiol. (2003) in press (10.1152/ajpcell.00255.2003.). [7] K.R. Gantt, U. Atasoy, R. Harrison, B. Cheatham, P.H. Pekala, HuR is a ligand for adipocyte mRNAs, GLUT1 & leptin, in: A. Ephrussi, I. Mattaj, G. Dreyfuss (Eds.), Proceedings of the Keystone Symposia on RNA Trafficking, Transport and Processing, Silverthorne, CO, 2003, p. 47.
HuB localizes to polysomes and alters C/EBP-b expression in 3T3-L1 adipocytes Kira R. Gantt, Renu G. Jain, Ronald W. Dudek, and Phillip H. Pekala* Department of Biochemistry and Molecular Biology, The Brody School of Medicine, Greenville, NC 27858, USA Received 26 November 2003
Abstract The RNA binding protein HuB was ectopically expressed in 3T3-L1 preadipocytes and localized primarily to the nucleus, as the cells differentiated HuB redistributed to the cytosol and on analysis localized to the dense polysomes. Electron micrographs confirm association of HuB with the ribosomes in the adipocytes consistent with a proposed role in control of translation and mRNA stability. Examination of the expression of C/EBP-b in the cells expressing HuB relative to the parental 3T3-L1 adipocytes demonstrated an alteration in the LAP to LIP ratio. The data support a role for endogenous Hu proteins in the differentiation process, potentially affecting the rate of differentiation by controlling the concentration of the dominant negative transcription inhibitor, LIP. Ó 2003 Elsevier Inc. All rights reserved. Keywords: 3T3-L1; Adipocytes; C/EBP-b; Differentiation; HuB; mRNA stability; Translation
HuB is an mRNA binding protein belonging to the RNA recognition motif family [1]. HuB binds to A + U rich elements (AREs) in the 30 UTR of various mRNAs controlling stability and translatability of the message [2,3]. In vitro RNA gel shifts demonstrated that an ARE in the 30 UTR of the GLUT1 mRNA was a ligand for HuB and ectopic expression of HuB in the 3T3-L1 cells led to an acceleration of the differentiation process and a 10-fold overexpression of the GLUT1 glucose transporter protein [2]. Interestingly, while both HuB and GLUT1 were expressed in the preadipocytes, no alteration of transporter expression occurred. This led us to propose that HuB remained sequestered in the nucleus prior to the initiation of differentiation whereupon it chaperoned mRNAs that may play a role in establishing and maintaining the adipocyte phenotype to the cytosol and controlled their expression. In the current study, we demonstrate that in the adipocytes, ectopically expressed HuB localizes to the ribosomes and alters expression of C/EBP-b relative to the parental 3T3-L1 cells. * Corresponding author. Fax: 1-252-744-3383. E-mail address: [email protected] (P.H. Pekala).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.162
Materials and methods Materials. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco/Invitrogen (Grand Island, New York). 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). All other chemicals were of reagent grade and purchased from Sigma– Aldrich Biochemical (St. Louis, MO). Antibody against the G10 tag (T7) was purchased from Novagen (Madison, WI). An isotype control IgG2 a was obtained from BD-Pharmingen (San Diego, CA). AuroProbe anti-mouse antibody labeled with 15 nm gold particles was purchased from Amersham (Piscataway, NJ). Antibodies directed against C/EBP-b and HuB were provided by M. Daniel Lane (The Johns Hopkins University, Baltimore, MD) and Jack Keene (Duke University, Durham, NC), respectively. 3T3-L1 cell culture and transfection. 3T3-L1 preadipocytes were cultured, maintained, and differentiated as previously described [2]. For experimentation preadipocytes were harvested at 2 days postconfluence and adipocytes were used at eight days postinduction of differentiation. Cells were transfected with pBC-HuB expression vector, selected for, and characterized as previously described [2]. The HuB cDNA contained a gene 10 (g10) epitope tag for immunoidentification. Polysome profiles: analysis of polysomes by sucrose density gradients. Polysome profiles were generated as described by Long and Pekala [4]. Western blot analysis. Western blot analysis was performed as previously detailed [5].
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Electron microscopy. HuB transfectants (preadipocytes or adipocytes) were collected by scraping, isolated by centrifugation (1600 rpm for 5 min), and fixed (4% formaldehyde, 0.1% glutaraldehyde in phosphate buffered saline, pH 7.4). The fixed cells were dehydrated by 10 min treatments with graded concentrations of ethanol (70%, 90%, 95%, and 100%), embedded in LR White plastic, and ultrathin sections were cut with a Reichter ultramicrotome (Reichter Otische Werke, Vienna, Austria). Immuno-cytochemistry was performed on sections using the monoclonal antibody to the T7 gene 10 epitope tag or an isotype control IgG2 a at 1:100 dilutions in the blotting solution. Secondary anti-mouse antibody conjugated with 15 nm gold particles was used for visualization. Blocking was accomplished with egg albumin. Sections were incubated in 2% OsO4 in 0.2 M phosphate buffer for 1 h and washed with phosphate buffered saline and then with distilled water. Sections were counterstained with 8% uranyl acetate in 50% ethanol and visualized with the JEOL 1200EX electron microscope operated at 60 kV. HuB nomenclature. HuB is the mammalian homolog of the Drosophilia embryonic lethal abnormal vision gene product and has been referred to in the previous literature as human embryonic lethal abnormal vision like neuronal protein 1 (Hel-N1). For the sake of consistency with contemporary literature we will refer to the gene and protein product as HuB.
Results and discussion Our previous studies demonstrated that ectopic expression of HuB altered the distribution of GLUT1 mRNA among ribosomes and polysomes, shifting the bulk of the message to the most dense fractions of the gradient. Remarkably, initiation complexes could not be detected, suggesting that HuB expression resulted in an acceleration of the initiation process and the loading of more ribosomes per polysome [2]. In the current study sucrose density gradient analysis was performed to determine if the ectopically expressed HuB localized to the same fractions within the 3T3-L1 adipocytes. The analysis was performed on day 8 following induction of differentiation and a representative polysome profile is presented in Fig. 1. In Fig. 1A, the profile depicts fractionation of the protein synthesis apparatus. Western blot analysis performed on protein isolated from selected fractions shown in Fig. 1B indicates that the bulk of the HuB protein can be found in the very dense fraction of the gradient. The distribution appears identical to that we have previously published for GLUT1 message in HuB expressing cells [2]. To further investigate the association of HuB protein with the ribosomes, electron microscopy was performed. As demonstrated in Fig. 2A, no immunogold staining was observed to be associated with the ribosomes in the preadipocytes. However, Fig. 2B, presenting samples derived from adipocytes, clearly demonstrates the colocalization of the immunogold and the ribosomes (see arrow). Thus, in the adipocytes, HuB co-sediments with dense polysomes in a sucrose gradient and can be visualized by electron microscopy as localized to ribosomal
Fig. 1. Polysome profile and HuB localization in 3T3-L1 adipocytes. (A) Polysome profile from HuB transfected cells. A postmitochondrial supernatant was prepared from HuB transfected cells on day 8 of a differentiation time course and applied to a 15–40% sucrose density gradient, which was centrifuged at 20,000 g for 2 h. The gradients were fractionated and the absorbance at 250 nm was measured generating a polysome profile. The top of the gradient is on the left. (B) HuB localization in polysome profile. Protein was extracted from the fractions and Western blot analysis was performed for HuB. Fraction numbers in the Western blot (B) correspond to fraction numbers in the polysome profile (A). The data presented are representative of duplicate determinations performed on each of two separate transfections.
structures, supporting a relationship between HuB and the translation apparatus. While our previous studies have focused on the effect of HuB on GLUT1 expression, we do not believe that the GLUT1 message is a ligand for HuB in isolation. Our model would suggest that HuB selects multiple mRNAs or perhaps families of related messages essential for establishing and maintaining the adipose phenotype, chaperones them to the cytosol, and controls their expression. Examination of differentiation related genes that might prove to be ligands for HuB yielded C/EBP-b. This transcription factor is expressed early in the differentiation program, with protein detected in the nucleus within 4 h of exposure of the cells to the differentiation stimuli [6]. Examination of the message demonstrates the presence of a strong ARE between bases 1291 and 1400 of the 30 UTR. If HuB did bind the C/EBP-b message, we would predict that much like GLUT1 an alteration in expression should be observed. Examination of C/EBP-b expression by Western blot analysis in the parental versus the HuB transfectants (Fig. 3) demonstrates that the HuB expressing cells display an altered expression of this transcription factor. Most notably is the alteration of the expression of the truncated form of C/EBP-b, LIP (Fig. 3B). As opposed
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Fig. 2. Localization of HuB by electron microscopy. Electron micrographs of HuB staining in preadipocyte and adipocyte cytosol. (A) Preadipocyte cytosol shows the presence of ribosomes (dark grey, triangular-shaped structures) arranged in coils, but no staining for HuB. Ribosomes are magnified in inset A. (B) Adipocyte cytosol shows the presence of HuB visualized with 15 nm gold particles closely associated with ribosomes. Inset B shows gold particles (black, rounded-shaped structures) superimposed on coiled ribosomes. Osmium/uranyl acetate staining was done to visualize cytosolic structures. The T7 gene 10 antibody was used to immuno-stain for HuB, with a goat anti-mouse 15 nm gold conjugate secondary. Use of an isotype control antibody showed no staining. Magnification: 25,000.
Fig. 3. Effect of HuB expression on C/EBP-b expression. (A) Cell protein extracts equivalent to 106 cells were prepared from parental 3T3-L1 cells during a differentiation time course and subjected to immunoblot analysis for C/EBP-b protein content as described in Materials and methods. The expression patterns for the full length form of the transcription factor C/EBP-b, also known as LAP (32 kDa) and the truncated dominant negative inhibitory form of C/EBP-b also known as LIP (21 kDa) are shown. (B) C/EBP-b expression in the HuB transfectants. The data are representative of duplicate determinations performed on each of two separate transfections.
Fig. 4. Effect of HuB expression on C/EBP-a expression. (A) Cell protein extracts equivalent to 106 cells were prepared from parental 3T3-L1 cells during a differentiation time course and subjected to immunoblot analysis for C/EBP-a protein content as described in Materials and methods. The expression patterns for the full length form of the transcription factor C/EBP-a, also known as 42 kDa and the truncated form 30 kDa are shown. (B) C/EBP-a expression in the HuB transfectants. The data are representative of duplicate determinations performed on each of two separate transfections.
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to the parental 3T3-L1 cells, LIP is expressed over a much shorter duration, detectable at day 0, maximum at day 4 with expression suppressed by day 7. This provides a significantly increased LAP (full length form) to LIP ratio in the HuB transfectants. This would suggest that HuB controls the LAP to LIP ratio through binding to the ARE and influencing translation. As LIP functions as a dominant negative C/EBP family member one might predict that due to the shorter duration of LIP expression and the LAP to LIP ratio, the differentiation process might be accelerated/enhanced in the HuB expressing cells. Indeed, examination of the expression of C/EBP-a (Fig. 4) confirms this prediction. In the 3T3-L1 parental cells (Fig. 4A), C/EBP-a expression is detected at day 5 with significant accumulation occurring between days 7 and 8. While in the HuB transfected cells (Fig. 4B), C/EBP-a expression can be detected at day 4 with significant accumulation by day 5. As C/EBP-b, at least in part, is thought to control expression of the C/ EBP-a gene, the earlier expression of the b-isoform and the increased LAP to LIP ratio in the HuB transfectants (Fig. 3) would be consistent with an earlier and robust expression of C/EBP-a. Finally, we note that examination of C/EBP-d expression indicated no differences between parental cells and the HuB transfectants (R. Jain and P.H. Pekala, unpublished results). We initiated experiments ectopically expressing HuB, a neuronal protein, in the 3T3-L1 cells [2] as previous attempts to transfect various mammalian cell lines with HuB resulted in cessation of cellular proliferation and/or dramatic morphological changes precluding analysis of specific effects of HuB. Those studies did indeed provided insight into the mechanism by which HuB controlled expression of its ligand mRNAs. We have since described the endogenous expression of another member of the Hu family in the adipocytes, HuR [7] and demonstrated using in vitro RNA gel shifts that the ARE contained in the 30 UTR of the C/EBP-b mRNA is a ligand for HuR (R. Tenney and P.H. Pekala, unpublished
results). Taken together these observations begin to provide insight into the function of HuR in adipocyte differentiation.
Acknowledgments The authors thank Jack Keene for thoughtful discussions of the work and acknowledge the expert technical assistance of Dan Whitehead in the performance of the electron microscopy and the grant support of the NIH (DK55769) and the American Diabetes Association.
References [1] T.D. Levine, F. Gao, P.H. King, L.G. Andrews, J.D. Keene, HelN1: an autoimmune RNA-binding protein with specificity for 30 urydilate-rich untranslated regions of growth factor mRNAs, Mol. Cell. Biol. 13 (1993) 3494–3504. [2] R.G. Jain, L.G. Andrews, McGowan, P.H. Pekala, J.D. Keene, Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes, Mol. Cell. Biol. 17 (1997) 954–962. [3] D. Antic, N. Lu, J.D. Keene, ELAV tumor antigen, Hel-N1 increases translation of neurofilament M mRNA and induces formation of neuritis in human teratocarcinoma cells, Genes Dev. 13 (1999) 449–461. [4] S. Long, P.H. Pekala, Regulation of GLUT4 mRNA stability by TNF-a: alterations in both protein binding to the 30 untranslated region and initiation of translation, Biochem. Biophys. Res. Commun. 220 (1996) 949–953. [5] J.M. Stephens, P.H. Pekala, Transcriptional repression of the C/EBPa and GLUT4 genes in the 3T3-L1adipocytes by tumor necrosis factor-a. Regulation is coordinate and independent of protein synthesis, J. Biol. Chem. 267 (1992) 13580–13584. [6] K.A. Cole, A.W. Harmon, J. Harp, Y.M. Patel, Rb regulates C/EBPb-DNA-binding activity during 3T3-L1 adipogenesis, Am. J. Cell. Physiol. (2003) in press (10.1152/ajpcell.00255.2003.). [7] K.R. Gantt, U. Atasoy, R. Harrison, B. Cheatham, P.H. Pekala, HuR is a ligand for adipocyte mRNAs, GLUT1 & leptin, in: A. Ephrussi, I. Mattaj, G. Dreyfuss (Eds.), Proceedings of the Keystone Symposia on RNA Trafficking, Transport and Processing, Silverthorne, CO, 2003, p. 47.