HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 26, 23563-23572, June 28, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/26/23563    most recent M202184200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parkin, S. E. Articles by Johnson, P. F. Search for Related Content PubMed PubMed Citation Articles by Parkin, S. E. Articles by Johnson, P. F. Social Bookmarking                      What's this?

Regulation of CCAAT/Enhancer-binding Protein (C/EBP) Activator Proteins by Heterodimerization with C/EBP (Ig/EBP)^*

Sara E. Parkin, Mark Baer, Terry D. Copeland^§, Richard C. Schwartz^¶, and Peter F. Johnson^**

From the  Eukaryotic Transcriptional Regulation Section, Regulation of Cell Growth Laboratory and the ^§ Basic Research Laboratory, NCI-Frederick, Frederick, Maryland 21702-1201 and the ^¶ Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1101

Received for publication, March 6, 2002, and in revised form, April 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The CCAAT/enhancer-binding proteins (C/EBPs) are basic leucine zipper transcription factors that play important roles in regulating cell growth and differentiation. C/EBP proteins form leucine zipper-mediated homodimers but are also capable of heterodimerizing with other C/EBPs in vitro. Here we show that C/EBP occurs predominantly as a heterodimer that displays rapid mobility in gel shift assays. Biochemical fractionation and antibody supershift assays demonstrate that the C/EBP heterodimeric partner is C/EBP (Ig/EBP), a C/EBP protein that has been implicated as an inhibitor of other family members. Although most cell types express C/EBP·C/EBP heterodimers, macrophages contain a C/EBP partner that is serologically distinct from C/EBP. We found that C/EBP blocked the ability of C/EBP and C/EBP to activate a reporter gene in L cell fibroblasts but did not inhibit a chimeric C/EBP protein containing the GCN4 leucine zipper. Repression by C/EBP occurs at the level of transactivation and requires heterodimerization with the C/EBP partner. C/EBP was an ineffective repressor in HepG2 hepatoma cells despite forming C/EBP heterodimers, and C/EBP was not effectively inhibited in either L or HepG2 cells. Our findings demonstrate that C/EBP modulates C/EBP activity in a cell- and isoform-specific manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Eukaryotic transcription factors commonly occur in families whose members share similar DNA binding specificities and other functional properties. Many transcription factors are dimeric and can form homodimers as well as heterodimers with other family members. The capacity to heterodimerize provides a means of enhancing regulatory diversity, as the various dimeric species within a protein family may exhibit distinct functional properties (1-4). Thus, it is important to elucidate the dimerization status of transcription factors in vivo to understand the full range of their biological activities and regulation.

In the present study, we have examined the dimerization properties of CCAAT/enhancer-binding proteins (C/EBPs)¹ in cells. C/EBPs are a family of basic leucine zipper (bZIP) DNA-binding proteins (5) consisting of five core members: C/EBP, C/EBP, C/EBP, C/EBP, and C/EBP (Ig/EBP). C/EBP proteins bind to DNA as dimers and display highly related DNA binding and dimerization specificities (reviewed in Refs. 6-8). C/EBPs are involved in the regulation of many cellular processes. C/EBP, C/EBP, and C/EBP mediate responses to stress and inflammatory signals, including regulation of acute phase response genes in hepatocytes and expression of proinflammatory cytokine genes in monocytic cells (9-13). Overexpression experiments and analysis of knockout mice demonstrate that C/EBP proteins also control cell growth and differentiation (6-8, 14). For example, forced expression of C/EBP and/or C/EBP in precursor cells of the adipocyte, granulocyte, and keratinocyte lineages causes growth arrest and induces cellular differentiation (15-17). In other contexts, C/EBP has been reported to stimulate cell growth (18, 19). Thus, C/EBPs regulate a variety of cellular phenotypes in a wide range of cell types.

In addition to forming homodimers, C/EBP proteins are capable of heterodimerizing with the other family members in vitro (20-22). Heterodimerization could potentially alter several functional activities of C/EBP proteins, including DNA binding, transactivation potential, responsiveness to signaling pathways, and the ability to cooperate with other transcription factors. It has been assumed that heterodimers between C/EBP family members occur in vivo and possess regulatory activities that are distinct from the homodimeric forms. However, there is only limited evidence for such heterodimers in vivo. An association between C/EBP and C/EBP was observed in transient overexpression experiments (20), and evidence has been reported for C/EBP·C/EBP heterodimers in liver nuclear extracts (23) and monocytic cells (24). In addition, C/EBPs appear to heterodimerize with proteins from other bZIP subfamilies, including Fos/Jun (25) and ATF/CREB (26-28).

Here we report that C/EBP proteins in cell and tissue extracts are found predominantly as heterodimers with C/EBP. C/EBP is a ubiquitously expressed member of the C/EBP family that was first identified by its affinity for cis-regulatory sites in the Ig heavy chain promoter and enhancer (29). C/EBP contains a C/EBP-like bZIP region but lacks an amino-terminal transactivation domain (30, 31) and can inhibit transcriptional activation by C/EBP or C/EBP (31). We show that C/EBP can repress C/EBP- and C/EBP-mediated transactivation of a reporter gene in fibroblasts in a leucine zipper-dependent manner, indicating that the repression by C/EBP involves heterodimerization with its partner. Interestingly, C/EBP did not repress transactivation by C/EBP or C/EBP in HepG2 hepatoma cells, nor did it inhibit C/EBP activity in either cell type. Thus, the ability of C/EBP to inhibit C/EBP activators is cell-specific and differs for the various C/EBP family members.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Cell Culture-- L cell fibroblasts were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Inc.) supplemented with 10% fetal bovine serum (HyClone, Inc.) in the presence of kanamycin, streptomycin, and penicillin. HepG2 hepatoma cells (ATCC HB-8065) were maintained in minimum essential Eagle's medium (BioWhittaker, Inc.) supplemented with nonessential amino acids, sodium pyruvate, 10% fetal bovine serum (HyClone, Inc.) in the presence of kanamycin, streptomycin, and penicillin. Other cell lines used for analysis of nuclear extracts were C6-2B (rat glioma; Ref. 32), IC-21 (murine macrophage; Ref. 33; ATCC TIB 186), and EMT6 (murine mammary tumor). P388-C and P388-C-C1 cells are stably transfected P388 lymphoblasts (ATCC CCL 46) expressing C/EBP and C/EBP, respectively (34, 35). P388 cell lines were grown in RPMI 1640 (BioWhittaker) supplemented with 10% fetal clone I serum (HyClone, Inc.), glutamine, kanamycin, streptomycin, and penicillin.

Antibodies-- C/EBP antiserum specific for the COOH terminus (C-19) was obtained from Santa Cruz Biotechnology. Peptide antisera recognizing the NH₂ terminus of C/EBP (20) and the NH₂ terminus of GCN4 (36) have been described. A polyclonal antiserum against bacterially expressed C/EBP (37) was kindly provided by K. Calame. Two peptide antisera were raised against murine C/EBP by immunizing rabbits with synthetic peptides corresponding to the amino-terminal (Ser-Lys-Leu-Ser-Gln-Pro-Ala-Thr-Thr-Pro-Gly-Val-Asn-Gly-Cys) or car boxyl-terminal peptide (Cys-Ile-Ser-Thr-Glu-Thr-Thr-Ala-Thr-Asn-Ser-Asp-Asn-Pro-Gly-Gln). Cysteine residues were included in both peptides for covalent coupling to carrier protein.

Plasmid Constructs-- The C/EBP coding sequence was amplified by PCR from a plasmid containing a C/EBP clone of murine origin (29). Two oligonucleotide primers were used, one overlapping the initiation codon with an introduced NcoI restriction site and the second spanning the termination codon with an introduced HindIII restriction site. The PCR product was digested with NcoI (partial) and HindIII (complete) and inserted into the pMEX expression vector to generate pMEX-C/EBP. A bacterial expression construct, pJL6-C/EBP, was generated by inserting the digested PCR product into the expression vector pJL6 as described (38). The GAL4-C/EBP hybrid construct was described previously (38).

Transient Transfections-- Transfections were carried out using 30-40% confluent monolayers in 10-cm dishes using FuGENE^TM (Roche Molecular Biochemicals). For co-transfection experiments, a constant amount of C/EBP reporter plasmid (DE1)₄-alb-luc (2.5 µg) (38) and C/EBP expression constructs pMEX-C/EBP, pMEX-C/EBP-G_LZ, pMEX-C/EBP, or pMEX-C/EBP (0.75 µg) were transfected with varying quantities of C/EBP expression construct pMEX-C/EBP (0.5-8.5 µg). pRSV--galactosidase (0.5 µg) was co-transfected to normalize for transfection efficiency. The total amount of DNA used for transfection (12.25 µg) was kept constant by adding an appropriate amount of the pMEX vector. After 48 h, the cells were lysed and analyzed for luciferase activity using the Enhanced Luciferase Assay Kit (PharMingen International) and for -galactosidase activity using the luminescent -galactosidase Genetic Reporter System II (CLONTECH Laboratories, Inc.).

GAL4-C/EBP transfection assays were conducted using 30-40% confluent monolayers in 60-mm wells using FuGENE^TM (Roche Molecular Biochemicals). One µg of (G)₅ E1B-luc reporter plasmid (38), 5 ng of GAL4-C/EBP vector, and 25 ng of Ha-Ras(12V) vector were transfected with varying quantities of the pcDNA3.1 C/EBP expression vector (13.3 ng to 1.25 µg). The Renilla luciferase vector, pRL-TK (Promega), was co-transfected as an internal standard for transfection efficiency. Sixteen hours prior to harvesting the cells, the medium was removed and replaced with serum-free media. Cells were collected 48 h after transfection, lysed, and analyzed using the Dual-Luciferase^® assay system (Promega).

Nuclear Extracts-- Nuclear extracts from cell lines and transfected cells were prepared by a detergent lysis method. Transfected cells were washed once with phosphate-buffered saline, scraped, and then divided. 20% of the cells from 10-cm dishes were used for luciferase assays by resuspension in detergent lysis solution (100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100, 1 mM dithiothreitol (DTT); CLONTECH Laboratories, Inc.), incubation at room temperature for 5 min and centrifugation. The remaining 80% of the cells were used to make nuclear extracts by resuspension in lysis buffer (20 mM HEPES (pH 7.9), 1 mM EDTA, 10 mM NaCl, 1 mM DTT, 0.1% (v/v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml antipain ) and incubation on ice for 10 min. Nuclei were pelleted by centrifugation at 3,500 rpm for 10 min. Proteins were extracted from nuclei by incubation in high salt buffer (25 mM HEPES (pH 7.9), 0.2 mM EDTA, 0.42 M NaCl, 0.2 mM DTT, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml antipain) at 4 °C for 20 min with vigorous shaking. Nuclear debris was pelleted by centrifugation at 14,000 rpm for 5 min, and the supernatant was collected and stored at 70 °C. Nuclear extracts from mouse tissues were prepared by an Nonidet P-40 lysis procedure as described previously (39).

Electrophoretic Mobility Shift Assay (EMSA)-- The following double-stranded oligonucleotides containing the wild-type consensus C/EBP site (bold) or a mutant C/EBP site (bold) were used as probes or competitor DNAs.

The probe was end-labeled using [³²P]dCTP and Klenow polymerase. DNA-binding assays were carried out in a 25-µl reaction containing 20 mM HEPES (pH 7.9), 200 mM NaCl, 5% (w/v) Ficoll, 5% (v/v) glycerol, 1 mM EDTA, 50 mM DTT, 0.01% Nonidet P-40, 0.06% bromphenol blue, 1.75 µg of poly(dI-dC), and 7.5 × 10⁴ cpm probe. After incubation for 20 min at room temperature, 10 µl of the binding reaction was loaded onto a 6% polyacrylamide gel in 1× TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) and electrophoresed at 160 V for 2 h. The gel was dried before autoradiography. Supershift assays were carried out by preincubating the nuclear extract with 2 µl of rabbit antiserum at 4 °C for 30 min before addition of the binding reaction mixture. Where appropriate, DNA-protein complexes were quantitated using a PhosphorImager and ImageQuant software (Molecular Dynamics). Mixing experiments to form C/EBP heterodimers were incubated at 45 °C for 20 min in the presence of 95 mM DTT.

Bacterially Expressed Proteins-- His-tagged C/EBP, C/EBP, C/EBP-G_LZ, and truncated C/EBP-(191-276) were expressed in Escherichia coli and purified as described (38).

Immunoprecipitation-- Approximately 5.8 µg of purified His-tagged C/EBP was incubated with 5.8 µg of His-tagged C/EBP or truncated C/EBP-(191-276) at 45 °C for 20 min in the following buffer: 0.2 M Tris (pH 7.5), 0.2 M NaCl, 5 mM EDTA, 10 mM DTT, 0.1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml antipain. 3 µl of NH₂-terminal C/EBP antiserum and 10 µl of protein A-Sepharose beads were then added. As a control, His-tagged C/EBP or truncated C/EBP-(191-276) were incubated alone in the same manner. After a 3-h incubation at 4 °C, the beads were collected by centrifugation and washed three times with buffer. 1× Laemmli sample buffer was added to the beads, and the samples were boiled for 5 min and centrifuged. The eluted proteins were resolved by SDS-PAGE and examined by Western blotting using Super Signal^® West HisProbe^TM kit (Pierce).

Immunoprecipitation experiments using nuclear extracts from transfected L or HepG2 cells were performed using the SeizeX immunoprecipitation kit (Pierce). Cells were transfected with 5 µg of control plasmid, 5 µg of tagged C/EBP vector, or 5 µg of tagged C/EBP vector with 5 µg of C/EBP vector, and nuclear extracts were prepared. Seventy-five µg of N.E. was added to protein A-Sepharose beads cross-linked to the NH₂-terminal GCN4 antibody and incubated for 3 h at 4 °C. The beads were washed three times with buffer and eluted with protein sample buffer and the eluted proteins analyzed by Western blotting.

Western Blotting-- Nuclear extracts were mixed with an equal volume of 2× sample buffer (40), heated at 95 °C for 5 min, and loaded on precast 12% or 16% SDS-PAGE gels (Novex). Gels were transferred to Immobilon-P membranes (Millipore) and blocked with 5% dry milk in Tris-buffered saline (pH 7.6). Blots were developed using the enhanced chemiluminescence (ECL) detection system (Pierce).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

C/EBP Occurs in Cells Predominantly as a Rapidly Migrating EMSA Complex-- To examine the dimeric state of C/EBP proteins in cells, we performed EMSA on a series of nuclear extracts using a consensus C/EBP binding site oligonucleotide as the probe. We initially focused our analysis on C/EBP, because this protein is widely expressed in cell lines and tissues. We examined nuclear extracts from cell lines representing glioma (C6-2B), mammary epithelia (EMT6), and macrophages (IC-21) (Fig. 1A). The cell line extracts contained multiple species that bound specifically to the C/EBP motif, as determined by the ability of the unlabeled wild-type probe, but not a mutated oligonucleotide, to compete for binding (Fig. 1B and data not shown). To identify complexes containing C/EBP, we performed supershift analysis using a C/EBP antibody. As shown in Fig. 1A, C/EBP binding activities were observed in all extracts, and the C/EBP EMSA complexes occurred in two forms. One complex exhibited the same mobility as a bacterially expressed C/EBP homodimer (lane 7), whereas a second species (indicated by asterisks) displayed considerably faster mobility in the gel. In all cases this faster migrating form was the predominant C/EBP species. We also analyzed extracts from P388-C cells, which express C/EBP from a retroviral vector (34, 35). P388 is a lymphoblastic tumor cell that contains very low levels of endogenous C/EBP proteins except C/EBP (Ref. 41; see below). P388-C nuclear extracts contained almost exclusively the rapidly migrating form of C/EBP (Fig. 1C, lane 2). Thus, ectopic C/EBP in P388 cells is detected predominantly as a high mobility EMSA species, similar to the results observed for endogenous C/EBP in the other cell lines (Fig. 1A).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   C/EBP proteins in cells occur primarily as rapidly migrating EMSA complexes. A, EMSA of nuclear extracts from cell lines. A consensus C/EBP binding site was used as the probe. Extracts (3-12 µg) were assayed in the presence or absence of the NH2-terminal C/EBP antibody. Positions of the rapidly migrating species are indicated by asterisks. Bacterially expressed C/EBP was analyzed in parallel (lane 7) to show the mobility of the C/EBP homodimer. B, specificity of C/EBP·DNA complexes. Increasing amounts of unlabeled C/EBP competitor probe (lanes 7-12) or a mutant oligonucleotide with a disrupted C/EBP site (lanes 1-6) were added to binding reactions containing nuclear extracts from L cells. C, EMSA of nuclear extracts from stably transfected P388 lymphoblasts. Extracts from P388 cells and a P388 transfectant expressing C/EBP (P388-C) were analyzed as described in panel A.

The Rapidly Migrating C/EBP Complexes Are Heterodimers-- The fact that the C/EBP antibody used for the supershift experiments recognizes the amino terminus of the protein indicates that the rapidly migrating complex contains an intact C/EBP subunit and is not a truncated isoform, such as LIP, that contains the COOH-terminal DNA-binding domain (42, 43). The absence of truncated forms of C/EBP in the extracts was confirmed by Western blotting using a COOH-terminal C/EBP antibody (data not shown). To test whether the rapidly migrating C/EBP complex is a heterodimer with another cellular protein, we performed a mixing experiment in which purified recombinant C/EBP was combined with nuclear extract from P388 cells and assayed by EMSA (Fig. 2). This procedure generated a rapidly migrating complex that co-migrated with the endogenous C/EBP complex from P388-C cells (compare lanes 1 and 4). Similar results were obtained using recombinant C/EBP and C/EBP (data not shown). In contrast, a chimeric C/EBP protein (C/EBP-G_LZ) containing a heterologous leucine zipper from the yeast GCN4 protein did not form the rapidly migrating complex with P388 extract (lanes 5 and 6). These findings show that the C/EBP leucine zipper is necessary to produce the rapidly migrating C/EBP complex and indicate that this species consists of a heterodimer between C/EBP and another nuclear protein.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Identification of a C/EBP heterodimerizing factor using an in vitro mixing assay. Recombinant C/EBP was assayed by EMSA either alone or after mixing with P388 cell nuclear extract. Nuclear extracts from P388-C cells or P388 cells are shown in lanes 1 and 2, respectively. Recombinant C/EBP-GLZ, which contains the GCN4 leucine zipper, did not form a rapidly migrating complex when mixed with P388 extract (lanes 5 and 6). The consensus C/EBP site oligonucleotide was used as the probe.

Identification of C/EBP as the C/EBP Heterodimeric Partner-- The rapid mobility of C/EBP heterodimers and their presence in all cell types examined indicate that the dimeric partner is a small, ubiquitously expressed protein. These features suggested that the partner might be C/EBP (Ig/EBP), a 16.4-kDa protein that can dimerize with other C/EBP proteins and is expressed at the mRNA level in many cell types (29, 31). A C/EBP polyclonal antiserum raised against recombinant C/EBP (31) did not supershift the C/EBP heterodimer, although it shifted a more rapidly migrating complex that corresponds to a C/EBP homodimer (data not shown). However, biochemical purification of the rapidly migrating complex to near homogeneity suggested that the heterodimerizing factor may indeed be C/EBP.² Therefore, we generated additional antisera using synthetic peptides corresponding to the C/EBP amino and carboxyl termini and tested the antibodies in EMSA supershift experiments. Both C/EBP antisera supershifted a partially purified, rapidly migrating C/EBP complex isolated from P388-C/EBP cells, as well as a putative C/EBP homodimer also present in this preparation (Fig. 3A). Thus, these two peptide antibodies recognize C/EBP complexes that appear to contain C/EBP.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   The rapidly migrating C/EBP heterodimers contain C/EBP. A, the C/EBP heterodimer reacts with two peptide antisera specific for C/EBP. A partially purified fraction containing the C/EBP heterodimer isolated from P388-C/EBP cells (data not shown) was analyzed by EMSA supershift using antisera raised against peptides corresponding to the amino or carboxyl termini of C/EBP (lanes 2 and 3, respectively). B, upper panel, nuclear extracts from the cell lines used in Fig. 1 were assayed by EMSA supershift using the COOH-terminal C/EBP antibody. P388-C-C1 is a P388 derivative expressing C/EBP. Positions of the C/EBP·C/EBP heterodimers and C/EBP homodimers are shown on the right. Lower panel, Western blot analysis of C/EBP in cell extracts. The nuclear extracts (~30 µg of protein) from the upper panel were analyzed by Western blotting using the COOH-terminal C/EBP antibody. Nuclear protein (1 µg) from untransfected (lane 6) or C/EBP-transfected (lane 7) L cells was analyzed in parallel to demonstrate that overexpressed C/EBP is identical to the endogenous protein. C, EMSA of C/EBP complexes in nuclear extracts from mouse tissues. Each extract was analyzed in the absence and presence of C/EBP and C/EBP antibodies. Bands corresponding to C/EBP·C/EBP heterodimers are indicated by asterisks. The last three lanes contain extracts from untransfected (lane 13), C/EBP-transfected (lane 14), and C/EBP- plus C/EBP-transfected (lane 15) L cells. The identities of the EMSA complexes are indicated on the right.

To determine whether the rapidly migrating C/EBP complexes in cell extracts are C/EBP heterodimers, we tested a panel of cell extracts in antibody supershift assays using the COOH-terminal C/EBP antibody. Fig. 3B (upper panel) shows that rapidly migrating C/EBP complexes from glioma cells (lanes 1 and 2) and mammary epithelial cells (lanes 3 and 4), as well as from P388-C cells (lanes 7 and 8), were supershifted by the C/EBP antibody. Similarly, a C/EBP heterodimeric complex from P388-C-C1 cells (lanes 9 and 10) reacted with the C/EBP antibody. Interestingly, the rapidly migrating C/EBP complex from IC-21 macrophages was only weakly supershifted by the COOH-terminal C/EBP antibody (lanes 5 and 6) and by the NH₂-terminal C/EBP antibody (data not shown), despite the fact that this complex migrates identically with C/EBP·C/EBP heterodimers from other cells. C/EBP complexes from two other monocyte/macrophage cell lines also did not react appreciably with C/EBP antibodies (data not shown). These findings indicate that a distinct, but functionally related, protein heterodimerizes with C/EBP in monocytic cells. Western blot experiments using the same panel of cell extracts (Fig. 3B, lower panel) confirmed that a protein of ~18 kDa, identical in size to ectopically expressed C/EBP (lane 9), was detected in all cells examined including macrophages.

Because our analysis of C/EBP dimerization thus far used transformed or immortalized cell lines, we next wished to determine whether C/EBP heterodimerizes with C/EBP in normal tissues. We prepared nuclear extracts from mouse liver, brain, ovary, and spleen and analyzed them by EMSA and antibody supershift experiments. As shown in Fig. 3C, each extract contained a rapidly migrating C/EBP complex (denoted by an asterisk) that could be supershifted by the C/EBP and C/EBP antibodies. Nuclear extracts from L cells transfected with C/EBP (lane 14) or C/EBP plus C/EBP (lane 15) were analyzed on the same gel to confirm the identities of the homo- and heterodimeric C/EBP and C/EBP EMSA species. In summary, the experiments of Fig. 3 show that C/EBP occurs mainly as a heterodimer with C/EBP in cell lines as well as animal tissues.

C/EBP Causes Cell-specific Repression of C/EBP-mediated Transcription-- We next investigated whether heterodimerization with C/EBP affects the transcriptional activity of C/EBP. Initially, we examined the effect of C/EBP on C/EBP-mediated transactivation using a C/EBP-dependent promoter-reporter construct ((DEI)₄-alb-Luc; Ref. 38) in HepG2 hepatoma cells (Fig. 4A, left panel). C/EBP alone increased reporter expression by ~15-fold. Co-transfecting increasing amounts of a C/EBP expression vector did not significantly diminish reporter gene expression, even at the highest dose of C/EBP (8.5 µg), which is an 11.3-fold excess of C/EBP over the C/EBP vector. This result was unexpected, because C/EBP was previously found to inhibit the transactivation function of C/EBP and C/EBP in B lymphoma cells, 3T3 fibroblasts, and promonocytic cells (31). Therefore, we performed a similar C/EBP titration experiment in L fibroblastic cells (Fig. 4A, right panel). In these cells C/EBP clearly inhibited C/EBP-mediated transactivation of the (DEI)₄-alb-Luc reporter in a dose-dependent manner. Luciferase activity decreased linearly with the amount of C/EBP vector added and reached 22% of the control level at the maximal dose of C/EBP. Similar results were obtained using a reporter construct containing two copies of a consensus C/EBP binding site (data not shown). Thus, C/EBP is capable of inhibiting C/EBP activity in L cells but not in HepG2 hepatoma cells.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   C/EBP inhibits C/EBP activity in a cell-dependent manner. A, HepG2 and L cells were cotransfected with a constant amount of a C/EBP reporter plasmid, (DE1)4-alb-luc (2.5 µg), pMEX-C/EBP expression vector (0.75 µg), and the indicated amounts of pMEX-C/EBP expression vector. The luciferase activity in cells transfected with the reporter and C/EBP alone was set to 100%. Luciferase data (dashed lines) represent the average (± S.E.) of at least three independent transfections. C/EBP binding activity (solid lines) was measured by quantitating the DNA-protein complexes in EMSAs (panel B) using a PhosphorImager. The indicated ratios of dimeric complexes were calculated and plotted. B, EMSA of nuclear extracts from the transfected cells. Positions of C/EBP homodimers (:), C/EBP·C/EBP heterodimers (:), and C/EBP homodimers (:) are shown. Quantitative analysis of the complexes is presented in panel A. C, Western blot analysis of C/EBP expression in the transfected cells. 20 µg of each nuclear extract was loaded onto the gel, and the blot was developed with a C/EBP antibody.

To assess the levels of homo- and heterodimeric C/EBP complexes in the transfected cells, we prepared nuclear extracts and subjected them to EMSA analysis (Fig. 4B). C/EBP homodimer and heterodimer levels were increased in the transfected cells. Heterodimers were observed in cells transfected with C/EBP alone, resulting from dimerization with endogenous C/EBP (lane 2). The amount of heterodimeric complex increased with the addition of C/EBP vector, as did the levels of C/EBP homodimer. The EMSA complexes were quantitated by phosphorimaging, and C/EBP homodimer levels were calculated either as the percentage of total C/EBP binding activity (heterodimer plus the two homodimeric species) or as the fraction of C/EBP binding activity (C/EBP homodimer plus heterodimer) (Fig. 4A). The proportion of C/EBP homodimer decreased with added C/EBP, and the dimerization curves were similar in HepG2 cells (no transcriptional repression by C/EBP) and L cells (repression). Western blotting showed that C/EBP did not alter C/EBP levels in the nuclear extracts (Fig. 4C). These experiments show that C/EBP·C/EBP heterodimers are formed in both cell types, suggesting that heterodimers are transcriptionally active in HepG2 cells but not in L cells.

To further examine whether inhibition of C/EBP by C/EBP requires heterodimerization, we used the zipper-swap mutant, C/EBP-G_LZ, which is unable to dimerize with C/EBP. Fig. 5A shows that C/EBP did not repress C/EBP-G_LZ activity in either HepG2 or L cells, whereas in HepG2 cells transactivation was actually enhanced at the highest dose of C/EBP. EMSA (Fig. 5B) verified that C/EBP-G_LZ·C/EBP heterodimers were not formed in the transfected cells, although homodimers were observed. Thus, the ability of C/EBP to inhibit C/EBP activity requires heterodimerization between the two proteins and is not the result of competitive binding of transcriptionally inactive C/EBP homodimers to the promoter.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Repression of C/EBP activity by C/EBP requires the C/EBP leucine zipper. A, co-transfection experiments were performed in HepG2 and L cells as described in Fig. 4, except that pMEX-C/EBP-GLZ was used instead of wild-type C/EBP. Data are the average (± S.E.) of three experiments. B, EMSA of nuclear extracts from the transfected cells. Positions of C/EBP-GLZ homodimers and C/EBP homodimers are indicated.

Because the DNA binding activity of C/EBP was not repressed by heterodimerization, it seemed likely that C/EBP inhibits transactivation. To investigate this possibility, we used a GAL4-C/EBP fusion protein whose ability to activate a UAS-dependent reporter gene (G₅E1b-luc) depends on the transactivation domain (TAD) of C/EBP (38). Normally the activity of full-length C/EBP fused to GAL4 is very low because of strong repression of the TAD by inhibitory sequences located in COOH-terminal regions of the molecule, including the bZIP domain (38, 44). However, GAL4-C/EBP can be activated by coexpression of oncogenic Ha-Ras.³ Therefore, we transfected GAL4-C/EBP with a Ha-Ras(12V) vector and the G₅E1b-luc reporter into L cells along with increasing amounts of the C/EBP vector. As shown in Fig. 6, C/EBP potently inhibited GAL4-C/EBP activity in a dose-responsive manner. This result demonstrates that heterodimerization with C/EBP suppresses the ability of the C/EBP TAD to stimulate transcription, even when C/EBP is tethered to DNA through a heterologous DNA-binding domain. C/EBP did not inhibit GAL4-C/EBP activity in HepG2 cells (data not shown), further supporting the observation that C/EBP repression is cell-specific.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   C/EBP inhibits transactivation by a GAL4-C/EBP fusion protein. L cells were transfected with the G5E1b-Luc reporter (1 µg), GAL4-C/EBP vector (5 ng), pcDNA3-Ha-Ras(12V) (25 ng), and the indicated amounts of pcDNA3.1-C/EBP. Luciferase data are the average (± S.E.) of three experiments.

We next asked whether C/EBP could repress transactivation by other C/EBP family members (Fig. 7). Neither C/EBP nor C/EBP was inhibited by C/EBP in HepG2 cells, and, in fact, C/EBP activity was stimulated nearly 2-fold at the maximal C/EBP dose. This enhanced activity was associated with increased expression of C/EBP (data not shown), the mechanism of which is unknown. Similar to C/EBP, C/EBP transactivation was repressed by C/EBP in L cells. In contrast, C/EBP activity was unaffected by C/EBP in L cells (Fig. 7) despite the fact that the two proteins formed heterodimers (data not shown). Collectively, our data indicate that C/EBP·C/EBP heterodimers are active in L cells, whereas C/EBP·C/EBP and C/EBP·C/EBP dimers are repressed.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Differential repression of the C/EBP family members by C/EBP. Inhibition of C/EBP- and C/EBP-mediated transactivation by C/EBP was analyzed in HepG2 and L cells, as described in Fig. 4. Repression of C/EBP activity is included for comparison (the data are reproduced from Fig. 4A). The data are the average (± S.E.) of three experiments.

DNA-independent Association of C/EBP and C/EBP in Vitro and in Vivo-- The observation that C/EBP proteins in cells occur predominantly as heterodimers with C/EBP suggested that they might preferentially dimerize with C/EBP. Using recombinant His-tagged C/EBP and C/EBP proteins, we performed coimmunoprecipitation assays to compare the ability of C/EBP to self-dimerize and to heterodimerize with C/EBP in vitro (Fig. 8A). Self-dimerization was examined by mixing full-length (p34) C/EBP with a truncated C/EBP protein (C/EBP-(192-276)) containing only the bZIP portion of the molecule. Mixtures of full-length C/EBP and either C/EBP or C/EBP-(192-276) were immunoprecipitated with an antibody directed against the amino terminus of C/EBP. The immunoprecipitates were analyzed by Western blotting using a reagent that detects the polyhistidine tag. In the absence of full-length C/EBP, neither of the other proteins was immunoprecipitated (lanes 1 and 2), as expected. When full-length C/EBP was added to the mixtures, C/EBP and C/EBP-(192-276) were detected in the precipitated fraction (lanes 3 and 4). Both proteins were immunoprecipitated with similar efficiency, suggesting that the C/EBP leucine zipper has comparable affinity for itself and for C/EBP. Thus, the predominance of heterodimers in vivo may be the result of a molar excess of C/EBP in cells, or, alternatively, dimerization might be regulated by a cellular mechanism such as phosphorylation.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   DNA-independent association of C/EBP and C/EBP in vitro and in vivo. A, purified His-tagged C/EBP, C/EBP, and truncated C/EBP-(192-276) (5.8 µg each) were mixed in the indicated combinations and subjected to immunoprecipitation using the NH2-terminal C/EBP antibody. The immunoprecipitates were analyzed by Western blotting using a His tag detection reagent. The individual purified proteins (0.36 µg each) are shown in lanes 5-7. B, association of C/EBP and C/EBP in transfected cells. Nuclear extracts (20 µg) from L and HepG2 cells transfected with the indicated expression vectors were analyzed for expression of C/EBP and epitope-tagged C/EBP by Western blotting (top two panels). The same nuclear extracts (75 µg) were subjected to immunoprecipitation using a GCN4 NH2-terminal antibody (bottom panel). The immunoprecipitates were analyzed for C/EBP by Western blotting using the COOH-terminal C/EBP antiserum.

We next examined the association between C/EBP and C/EBP in transfected cells using a co-immunoprecipitation assay. Epitope-tagged C/EBP, which contains the NH₂-terminal 13 amino acids of GCN4 fused to its amino terminus, was expressed in L or HepG2 cells, either alone or with C/EBP. Nuclear extracts were prepared and subjected to immunoprecipitation with an antibody recognizing the GCN4 tag (36), followed by Western blotting for C/EBP. As shown in Fig. 8B (bottom panel), ectopic C/EBP co-immunoprecipitated with C/EBP in L cells and in HepG2 cells (lanes 3 and 6). Thus, in both cell types C/EBP and C/EBP are associated in the absence of DNA. These findings further support the conclusion that impaired heterodimerization does not explain the inability of C/EBP to repress transcription in HepG2 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our studies demonstrate that C/EBP activator proteins exist predominantly as heterodimers with C/EBP in vivo. By comparing EMSA complexes generated with recombinant C/EBP with those from nuclear extracts, we observed that C/EBP in cell lines and tissues occurs mainly as a rapidly migrating heterodimer. Antibodies specific for the NH₂ and COOH termini of C/EBP supershifted the rapidly migrating C/EBP species, confirming that the heterodimers contain C/EBP. Our characterization of C/EBP heterodimers in this study has focused on C/EBP, because this isoform is expressed in many cell lines. However, C/EBP and C/EBP also occurred as rapidly migrating heterodimers in P388 transfectants that stably express these proteins (Fig. 3B and data not shown), as well as in transiently transfected L cells (data not shown). Thus, it seems likely that all of the C/EBP activators heterodimerize with C/EBP in vivo.

Heterodimerization with C/EBP did not detectably alter the DNA-binding specificity of its C/EBP partner, because homodimers and heterodimers bound efficiently to a consensus C/EBP element. The major effect of dimerization with C/EBP was to repress C/EBP transactivation function. In L cells, coexpression of C/EBP inhibited the ability of C/EBP and C/EBP to activate transcription from a C/EBP-dependent promoter. A C/EBP chimera containing the GCN4 leucine zipper that cannot heterodimerize with C/EBP was resistant to repression. C/EBP also suppressed transactivation by a GAL4-C/EBP fusion protein. Collectively, these results indicate that heterodimerization with C/EBP inhibits the transcriptional activity of C/EBP. At present it is unclear how heterodimerization with C/EBP suppresses transactivation. C/EBP lacks a TAD and by itself neither activates nor represses transcription of target genes (31).² It is possible that, because C/EBP heterodimers contain only one activating subunit, they cannot efficiently stimulate transcription. Alternatively, heterodimerization with C/EBP might block access to a coactivator protein for which association with the C/EBP activator involves sequences in the leucine zipper and/or basic region. This possibility is currently under investigation.

The fact that C/EBP did not repress transactivation by any of the C/EBPs in HepG2 hepatoma cells is noteworthy. Analysis of the C/EBP dimeric species expressed in transfected cells showed that heterodimers were produced and their levels increased in proportion to the amount of transfected C/EBP vector. A homodimeric C/EBP complex was observed in both L and HepG2 cells, and this complex did not appreciably diminish with increased C/EBP expression (Fig. 4B). It is possible that a pool of homodimers exists that is resistant to heterodimerization, perhaps because of a specific post-translational modification. However, because the occurrence of these persistent homodimers did not differ in the two cell types, their presence cannot account for the differential repression by C/EBP.

Because there was no difference in heterodimer formation in HepG2 and L cells, at least as assessed by EMSA and co-immunoprecipitation experiments, we postulate that C/EBP·C/EBP heterodimers are transcriptionally active in HepG2 cells but not in L cells. There are several potential explanations for this difference in activity. Heterodimers could be the target of activating kinases in HepG2 cells but not in L cells, whereas homodimers might be effective substrates in both cells. Such modifications could occur on either the C/EBP activator protein or the C/EBP subunit. It is also conceivable that protein:protein interactions mediated by the bZIP region are necessary for transcriptional activation and that heterodimeric bZIP domains are differentially active for this function in the two cell types. Irrespective of the mechanism, the ability of C/EBP to affect the transactivation potential of C/EBP activators in a cell-specific manner represents a novel means of controlling C/EBP activity.

A mouse strain carrying a null mutation at the C/EBP locus has been developed (45). Homozygous mutant animals show grossly normal embryonic development and are initially viable after birth. However, the majority of mutant mice die within 48 h of postnatal development. Although the cause of mortality was not determined, the lethal phenotype shows that C/EBP has an essential function in newborn animals and presumably also in adult mice. It remains to be determined whether the lethality of C/EBP-deficient mice results from the absence of C/EBP heterodimers, leading to formation of homodimers with altered regulatory activities. Considering the involvement of C/EBP proteins in many biological processes and the predominance of C/EBP·C/EBP heterodimers in cells, it is not surprising that deletion of C/EBP would have severe phenotypic consequences. An additional function for C/EBP in lymphoid cells was revealed by analysis of bone marrow chimeras generated from C/EBP null donor cells (45). Natural killer cells derived from mutant donors display reduced cytolytic activity and impaired production of interferon- in response to interleukin-12 or interleukin-18 stimulation. Nevertheless, the molecular basis for defective interferon- gene expression in C/EBP-deficient natural killer cells has not been elucidated.

In another study examining C/EBP function in vivo, Zafarana et al. (46) created transgenic mice overexpressing C/EBP in erythroid cells. Animals heterozygous for the C/EBP transgene displayed increased fetal -globin gene expression compared with adult -globin expression, indicating that C/EBP positively regulates -globin transcription. However, when C/EBP expression was increased further by making the transgenic allele homozygous, fetal erythropoiesis was eliminated and the embryos did not survive beyond embryonic day 14.5. These results demonstrate that C/EBP stoichiometry critically affects development of the erythroid lineage. We suggest that the developmental defects associated with high ectopic C/EBP expression may result from decreased levels of C/EBP homodimers in erythroid precursor cells.

In addition to regulating transcription, C/EBP proteins can induce cell growth arrest (47-49). In proliferating cell lines, C/EBP proteins occur primarily as heterodimers, raising the possibility that heterodimerization with C/EBP mitigates the growth arrest activity of these proteins. In experiments to create P388 cell lines expressing the zipper swap mutant, C/EBP-G_LZ, only minimal amounts of the mutant protein were detected in stable transfectants whereas the wild type protein could be expressed at much higher levels (41). This result is consistent with the idea that C/EBP must heterodimerize with C/EBP for its expression to be tolerated in proliferating cells. Furthermore, HepG2 hepatoma cells express significantly lower levels of C/EBP and C/EBP than are found in normal, terminally differentiated hepatocytes (50). We speculate that C/EBP may be unable to suppress C/EBP-mediated growth arrest in hepatoma cells, similar to its inability to inhibit C/EBP-dependent transcription in these cells. Thus, conversion of hepatocytes to proliferating hepatoma cells might require strong down-regulation of C/EBP and C/EBP expression. In future studies it will be informative to examine the ability of C/EBP to modulate C/EBP-mediated cell growth arrest in various cellular contexts.

The observation that C/EBP activity can be inhibited by heterodimerization with C/EBP suggests that C/EBP dimerization might be regulated to control gene transcription. In this regard, calcium-regulated phosphorylation of a serine residue in the leucine zipper of C/EBP has been linked to its increased transcriptional activity (51). Although the molecular mechanism underlying this activation event has not been elucidated, the authors raised the possibility that phosphorylation of the C/EBP zipper might control dimerization. Our studies indicate that C/EBP could be involved in this putative regulatory mechanism. Although our experiments thus far have focused on artificial promoters, future studies will address potential differences between C/EBP homodimers and heterodimers in activating authentic promoters, in addition to the possibility that C/EBP heterodimerization is regulated by developmental cues or other physiological signals.

	ACKNOWLEDGEMENTS

We thank Kathryn Calame for providing C/EBP antiserum and Vicky Heath and Esta Sterneck for critical comments on the manuscript.

	FOOTNOTES

^* 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.

Supported by Grant-in-aid 9950490N from the American Heart Association.

^** To whom correspondence should be addressed: Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, MD 21702-1201. Tel.: 301-846-1627; Fax: 301-846-5991; E-mail: johnsopf@ncifcrf.gov.

Published, JBC Papers in Press, April 29, 2002, DOI 10.1074/jbc.M202184200

² S. Parkin and P. F. Johnson, unpublished results.

³ J. D. Shuman and P. F. Johnson, unpublished results.

	ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; bZIP, basic region leucine zipper; EMSA, electrophoretic mobility shift assay; TAD, transactivation domain; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. Sebastian and P. F. Johnson RasV12-Mediated Down-regulation of CCAAT/Enhancer Binding Protein {beta} in Immortalized Fibroblasts Requires Loss of p19Arf and Facilitates Bypass of Oncogene-Induced Senescence Cancer Res., March 15, 2009; 69(6): 2588 - 2598. [Abstract] [Full Text] [PDF]

				T. Akasaka, T. Balasas, L. J. Russell, K.-j. Sugimoto, A. Majid, R. Walewska, E. L. Karran, D. G. Brown, K. Cain, L. Harder, et al. Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) Blood, April 15, 2007; 109(8): 3451 - 3461. [Abstract] [Full Text] [PDF]

				T. T. C. Yang, P. M. U. Ung, M. Rincon, and C.-W. Chow Role of the CCAAT/Enhancer-binding Protein NFATc2 Transcription Factor Cascade in the Induction of Secretory Phospholipase A2 J. Biol. Chem., April 28, 2006; 281(17): 11541 - 11552. [Abstract] [Full Text] [PDF]

				P. F. Johnson Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors J. Cell Sci., June 15, 2005; 118(12): 2545 - 2555. [Abstract] [Full Text] [PDF]

				A. D. Burkart, A. Mukherjee, E. Sterneck, P. F. Johnson, and K. E. Mayo Repression of the Inhibin {alpha}-Subunit Gene by the Transcription Factor CCAAT/Enhancer-Binding Protein-{beta} Endocrinology, April 1, 2005; 146(4): 1909 - 1921. [Abstract] [Full Text] [PDF]

				K. Hardy, L. Mansfield, A. Mackay, S. Benvenuti, S. Ismail, P. Arora, M. J. O'Hare, and P. S. Jat Transcriptional Networks and Cellular Senescence in Human Mammary Fibroblasts Mol. Biol. Cell, February 1, 2005; 16(2): 943 - 953. [Abstract] [Full Text] [PDF]

				V. Heath, H. C. Suh, M. Holman, K. Renn, J. M. Gooya, S. Parkin, K. D. Klarmann, M. Ortiz, P. Johnson, and J. Keller C/EBP{alpha} deficiency results in hyperproliferation of hematopoietic progenitor cells and disrupts macrophage development in vitro and in vivo Blood, September 15, 2004; 104(6): 1639 - 1647. [Abstract] [Full Text] [PDF]

				M. Roth, P. R.A. Johnson, P. Borger, M. P. Bihl, J. J. Rudiger, G. G. King, Q. Ge, K. Hostettler, J. K. Burgess, J. L. Black, et al. Dysfunctional Interaction of C/EBP{alpha} and the Glucocorticoid Receptor in Asthmatic Bronchial Smooth-Muscle Cells N. Engl. J. Med., August 5, 2004; 351(6): 560 - 574. [Abstract] [Full Text] [PDF]

				H. Schrem, J. Klempnauer, and J. Borlak Liver-Enriched Transcription Factors in Liver Function and Development. Part II: the C/EBPs and D Site-Binding Protein in Cell Cycle Control, Carcinogenesis, Circadian Gene Regulation, Liver Regeneration, Apoptosis, and Liver-Specific Gene Regulation Pharmacol. Rev., June 1, 2004; 56(2): 291 - 330. [Abstract] [Full Text] [PDF]

				P.-P. Kuang and R. H. Goldstein Regulation of elastin gene transcription by interleukin-1{beta}-induced C/EBP{beta} isoforms Am J Physiol Cell Physiol, December 1, 2003; 285(6): C1349 - C1355. [Abstract] [Full Text] [PDF]

				M. D. Bjerregaard, J. Jurlander, P. Klausen, N. Borregaard, and J. B. Cowland The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow Blood, June 1, 2003; 101(11): 4322 - 4332. [Abstract] [Full Text] [PDF]

				T. T. C. Yang and C.-W. Chow Transcription Cooperation by NFAT{middle dot}C/EBP Composite Enhancer Complex J. Biol. Chem., April 25, 2003; 278(18): 15874 - 15885. [Abstract] [Full Text] [PDF]

				H. Gao, S. Parkin, P. F. Johnson, and R. C. Schwartz C/EBPgamma Has a Stimulatory Role on the IL-6 and IL-8 Promoters J. Biol. Chem., October 4, 2002; 277(41): 38827 - 38837. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/26/23563    most recent M202184200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parkin, S. E. Articles by Johnson, P. F. Search for Related Content PubMed PubMed Citation Articles by Parkin, S. E. Articles by Johnson, P. F. Social Bookmarking                      What's this?