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INTRODUCTION |
The cornified envelope is a covalently cross-linked layer of
protein that is formed by epidermal keratinocytes during the final
stages in differentiation (1, 2). Involucrin
(hINV)1 is a
precursor of the keratinocyte cornified envelope that functions as a
glutamyl donor and amine acceptor in the
transglutaminase-dependent cross-linking reaction (1,
3-7). For proper envelope formation the transglutaminase enzyme and
its substrates (the envelope precursors) must be expressed at the
appropriate time and level during the differentiation process. Abnormal
expression or lack of expression can result in disease (8-10).
Involucrin is exclusively expressed in the suprabasal epidermal layers
(1, 7, 11-13). The mechanisms that regulate hINV expression during
keratinocyte differentiation are an area of active investigation
(14-22). The proximal regulatory region (PRR) of the hINV promoter is
located between positions
241 and
7 relative to the start of
transcription (20, 21). The PRR drives nearly one-half of the activity
of the promoter. Site specific mutation experiments indicate that an
activator protein 1 (AP1)-binding site, AP1-1, located within the PRR,
is absolutely required for promoter activity (20). This site interacts
with JunB, JunD, and Fra-1 (20). In addition to the AP1-1 site, this
region contains a C/EBP site that is necessary for promoter activity
(14).
C/EBP factors comprise a family of related bZIP (basic region leucine
zipper) DNA-binding proteins that regulate transcription. This family
includes C/EBP
, C/EBP
, C/EBP
, GADD153, CHOP, and LAP (23-28).
C/EBP factors have been shown to differentially modulate transcription
and differentiation in adipocytes, myelomonocytic cells, and ovarian
follicles (24, 26, 29, 30). Based on previous studies showing that the
C/EBP-binding site of the hINV promoter is important for activity (14),
we hypothesized that C/EBP factors may have a role in regulating hINV
expression during keratinocyte differentiation. In the present study we
show that each C/EBP protein differentially regulates hINV promoter
activity via the hINV promoter C/EBP site and that C/EBP factor binding to this site is dramatically increased following treatment of keratinocytes with 12-O-tetradecanoylphorbol-13-acetate
(TPA). We show that C/EBP
is a component of this complex. We also
use an inhibitory member of the C/EBP family, GADD153, to show that C/EBP factor activity is required for the TPA-dependent
increase in activity.
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MATERIALS AND METHODS |
Reagents--
[
-32P]ATP (3000 µCi/mmol) was
purchased from NEN Life Science Products. Keratinocyte serum-free
medium (KSFM), trypsin, Hanks' balanced salt solution, gentamicin, and
Lipofectin were obtained from Life Technologies, Inc. The pGL2 plasmid
and the chemiluminescent luciferase assay systems were obtained from
Promega. Phorbol ester (TPA) and dimethyl sulfoxide (Me2SO)
were obtained from Sigma. Chemiluminescence was measured using a
Berthold luminometer, and synthetic oligonucleotides were synthesized
using an Applied Biosystems DNA synthesizer.
Tissue Culture--
Human foreskin keratinocytes were isolated
and cultured as described (20, 21). The cells were passaged at a split
ratio of 1:5 when 70% confluent and used for transfection at the third passage.
Plasmid Construction--
The structure of the hINV promoter
reporter plasmid, pINV-241, has been described (20). To create the
C/EBP site mutant, the fragment containing the wild type C/EBP site
(5'-GCTGCTTAAG-3') was released as part of a larger fragment by
digestion of pINV-241 with ApaI/PstI and replaced
with the identical segment containing a mutated C/EBP site
(5'-GCTGAGATCT-3'). The modified nucleotides are
underlined. The structure of the mutated AP1-1 site in pINV-241(AP1-1m) has been previously described (20). The junction between the hINV gene
sequences and the luciferase reporter gene sequence is identical in all constructs.
Cell Transfection and Luciferase Assay--
Keratinocytes (60%
confluent) were transfected in 60-mm-diameter dishes. Lipofectin
reagent (16 µg) and 4.0 µg of test plasmid were added to cells in 3 ml of KSFM and incubated for 5 h at 37 °C. After 5 h,
additional KSFM (3 ml) was added, and the incubation was continued for
another 19 h. After 24 h in fresh KSFM, cells were washed and
treated for 24 h with KSFM or KSFM containing 50 ng/ml TPA
(delivered from a 5 mg/ml stock in Me2SO) (20, 21). Control
groups received the Me2SO vehicle. For cotransfection experiments, involucrin reporter plasmid (2.5 µg) was transfected with various concentrations of transcription factor expression plasmid.
The final expression vector concentration was maintained constant by
addition of an empty expression vector. The C/EBP factors C/EBP
(CRP2, rat) and C/EBP
(CRP3, mouse) were expressed using pMEX (Dr.
Peter Johnson of the Frederick Cancer Research Center) (25). C/EBP
was obtained from Dr. David Samols (Case Western Reserve University)
(31). GADD153 and pCMV-neo were obtained from by Dr. Nikki
Holbrook (27, 28). The cells were harvested and assayed for luciferase
activity as outlined above. For luciferase assay, cells were washed
twice with phosphate-buffered saline, dissolved in 250 µl of cell
culture lysis reagent (Promega), and harvested by scraping. Luciferase
assays were performed immediately using a Promega luciferase assay kit
(20). The results are expressed as luciferase activity per µg of
protein. All assays were performed in triplicate, and each experiment
was repeated a minimum of three times. The triplicates routinely varied
by less than 15%. As a control to assure comparable transfection
efficiency, we utilized a green fluorescent protein plasmid and
determined the percent of cells transfected by visual inspection (21,
32).
Gel Mobility Shift Assay--
For mobility shift assays, the
reaction (20 µl) contained 15% glycerol, 75 mM KCl,
0.375 mM dithiothreitol, 0.375 mM
phenylmethylsulfonyl fluoride, 12.5 mM NaCl, 0.1 mg/ml
poly(dI-dC), 2.5 µg of nuclear extract, and 0.3 ng of radiolabeled
DNA. The mixture was incubated for 5 min at room temperature, and
samples were immediately electrophoresed at 250 V for 1.5 h on a
5% nondenaturing acrylamide gel using 0.25× TBE running buffer. The
gels were then dried for autoradiography. For competition studies,
radioinert DNA competitor was added as a 20- or 200-fold molar excess.
For gel supershift assays, the complete gel mobility shift assay
mixture, without the 32P-labeled oligonucleotide, was
incubated at 4 °C for 2 h in the presence of an antibody
specific for the C/EBP isoform using 1 µg of rabbit IgG per reaction.
Dr. Steven McKnight kindly provided the C/EBP
-specific rabbit
polyclonal antibodies generated against amino acids 247-358 of rat
C/EBP
(C/EBP
-(247-358)). An additional C/EBP
-specific
antibody was obtained from Santa Cruz Biologicals (catalog no. sc-61X).
The 32P-labeled DNA was then added to the mixture and
incubated at room temperature for 5 min. The resulting complexes were
electrophoresed on a nondenaturing gel for characterization. C/EBP
(sc-150)- and C/EBP
(sc-636)-specific antibodies were obtained from
Santa Cruz Biologicals.
Immunological Detection of C/EBP
--
Cultured keratinocytes,
grown in KSFM, were treated for 24 h with KSFM or with KSFM
containing 50 ng/ml TPA before preparation of nuclear extracts as
described previously (20, 33). Equal quantities of nuclear protein were
electrophoresed on a 10% denaturing polyacrylamide gel and transferred
to nitrocellulose. C/EBP
was detected using rabbit anti-human
C/EBP
at a dilution of 1:500 (catalog no. sc-61X, Santa Cruz
Biologicals) followed by a goat anti-rabbit IgG secondary antibody used
at a 1:10,000 dilution. Secondary antibody binding was visualized using
chemiluminescent detection reagents.
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RESULTS |
hINV Promoter C/EBP Site Is Necessary for TPA-dependent
Regulation--
A C/EBP transcription factor binding site located in
the hINV promoter proximal regulatory region is required for hINV
promoter activity (14). Fig. 1 shows that
basal promoter activity (open bars) is reduced to 5-10% of
control in the absence of a functional C/EBP site. In addition, TPA
treatment increases promoter activity 8-fold; this
TPA-dependent activation is absent when the C/EBP site is
mutated (solid bars). These results demonstrate a
requirement for the C/EBP site for both basal and TPA-activated
promoter activity.

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Fig. 1.
Role for the hINV C/EBP-binding site.
Keratinocytes were grown until 60% confluent and then transfected with
2.5 µg of pINV-241 or pINV-241(C/EBPm) with or without treatment with
50 ng/ml TPA (see "Materials and Methods"). The cells were then
harvested, and extracts were prepared and assayed for luciferase
activity. The sequences of the intact and mutant C/EBP sites are listed
under "Materials and Methods." Each data point represents the
average value based on three separate experiments, and the
bars show the standard error of the mean.
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C/EBP Proteins Regulate Promoter Activity--
We next tested the
effects of C/EBP transcription factors on promoter activity. Fig.
2A shows that transfection of
increasing concentrations of C/EBP
expression plasmid with a
constant amount of hINV reporter plasmid increases hINV promoter
activity in a concentration-dependent manner in the absence of
TPA treatment. Expression is maximally increased by 0.4 µg of
C/EBP
plasmid, and the level is not significantly increased at
higher concentrations. As different C/EBP heterodimers are known to
differentially regulate gene expression, we determined whether C/EBP
and C/EBP
can regulate the C/EBP
-dependent
activation. As shown in Fig. 2B, C/EBP
and -
are
equally efficient inhibitors of the C/EBP
-dependent
activity. In addition, as shown in Fig.
3, the C/EBP proteins do not regulate promoter activity when the C/EBP-binding site is mutated. These results
(i) show that C/EBP proteins can regulate hINV promoter activity in the
absence of TPA treatment, (ii) provide evidence for a dynamic
regulatory interaction among C/EBP proteins, and (iii) demonstrate that
the proteins act through the C/EBP-binding site.

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Fig. 2.
C/EBP factors regulate hINV promoter
activity. Keratinocytes were transfected with pINV-241 (2.5 µg)
and increasing concentrations of C/EBP expression plasmid
(A). The expression vector plasmid concentration in all
transfections was maintained constant by the addition of empty
expression vector. In panel B, keratinocytes were
transfected with 2.5 µg of pINV-241, 0.5 µg of C/EBP , and
increasing concentrations of C/EBP or C/EBP . After an appropriate
incubation period (see "Materials and Methods"), the cells were
harvested and assayed for luciferase activity. The values represent the
mean determined from three individual experiments. Standard error
bars are shown.
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Fig. 3.
C/EBP factor regulation requires the hINV
C/EBP site. Keratinocytes were transfected with 2.5 µg of
pINV-241 or pINV-241(C/EBPm) in the presence of 2.5 µg of empty
expression vector, pMEX (C, control), or vector expressing
C/EBP , C/EBP , or C/EBP . After transfection, the cells were
harvested, and extracts were prepared and assayed for luciferase
activity as outlined under "Materials and Methods." The values are
the average of four independent experiments ± S.E.
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GADD153 Inhibits C/EBP
and TPA-dependent Promoter
Activity--
To obtain additional evidence of a role for C/EBP
factors in TPA-dependent activation, we treated
keratinocytes with TPA in the presence of GADD153, a C/EBP family
member that inhibits the activity of other C/EBP proteins by inhibiting
interaction of the C/EBP-GADD153 complex with DNA (23, 27). This
treatment creates an environment in which C/EBP activity is selectively eliminated. As shown in Fig.
4A, transfection of
TPA-treated cells with increasing concentrations of GADD153 produces a
concentration-dependent inhibition of
TPA-dependent promoter activity. As shown in Fig. 2,
C/EBP
activates the promoter in the absence of TPA treatment. We
would predict that GADD153 would also inhibit the
C/EBP
-dependent activation. Fig. 4B shows
that GADD153 produces a concentration-dependent inhibition
of C/EBP
-dependent activation of promoter activity. In
addition, GADD153 produced a concentration-dependent
inhibition of basal promoter activity; 0.4 µg of GADD153 expression
plasmid/transfection reduced basal activity to 5% of normal (not
shown).

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Fig. 4.
GADD153 inhibits TPA- and
C/EBP -dependent promoter
activity. Keratinocytes were cotransfected with pINV-241 (2.5 µg) and increasing concentrations of pGADD153 and then treated with
50 ng/ml TPA (A). B, cells were cotransfected
with 2.5 µg of pINV-241 + 0.5 µg of pC/EBP and increasing
concentrations of pGADD153. The cells were then harvested and assayed
for luciferase activity. The values are the average of three separate
experiments ± S.E.
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TPA Treatment Increases Binding to the C/EBP Site--
The above
described results provide functional evidence suggesting a role for
C/EBP proteins as regulators of hINVgene expression. To determine
whether TPA treatment alters C/EBP binding, we treated cultures with or
without TPA and assayed binding by gel mobility shift assay using the
double-stranded hINV C/EBP site oligonucleotide, 5'-GGTTTGCTGCTTAAGATGCCTG-3' (C/EBP-binding site in bold). As shown in Fig. 5, gel mobility shift
assay indicates that binding to 32P-labeled C/EBP-binding
site is increased by >10-fold following treatment with TPA
(lanes 1 and 2). The binding is specific, because addition of a 20- or 200-fold molar excess of radioinert C/EBP oligonucleotide inhibits the binding (lanes 3 and
4). No inhibition of binding to 32P-labeled
C/EBP oligonucleotide is observed with oligonucleotides encoding
consensus AP2 or ets binding sites or a mutant C/EBP-binding site (not
shown). In addition, two different C/EBP
-specific antibodies identify the complex prepared from TPA-treated cells as containing C/EBP
(lanes 5 and 6). C/EBP
- or
C/EBP
-specific antibodies do not detect protein binding (not
shown).

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Fig. 5.
Gel mobility shift analysis of C/EBP-binding
site. Nuclear extracts were prepared from near confluence
keratinocyte cultures following a 24-h treatment with or without 50 ng/ml TPA. The extracts were then incubated with
32P-labeled double-stranded oligomer,
5'-GGTTTGCTGCTTAAGATGCCTG-3', containing the hINV
C/EBP-binding site. Specificity was demonstrated by including a 20- and
200-fold molar excess of homologous competitor (comp) during
the binding reaction. The reactions shown in the two lanes on the
right were incubated with two distinct C/EBP -specific
antisera. Complexes were separated by electrophoresis and visualized by
autoradiography. FP indicates free probe, and the
lower arrowhead indicates C/EBP binding. The
arrowhead with the asterisk indicates the
supershifted C/EBP .
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TPA Treatment Does Not Increase C/EBP
Level--
The gel
mobility shift experiment indicate that C/EBP
binding to the hINV
C/EBP site is increased by TPA treatment. To determine whether this is
caused by a change in C/EBP
level, we measured C/EBP
levels in
nuclear extracts prepared from untreated and TPA-treated keratinocytes.
As shown in Fig. 6, TPA treatment did not
increase C/EBP
protein levels.

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Fig. 6.
Detection of C/EBP
in keratinocyte nuclear extracts. Nuclear extracts were
prepared from keratinocytes after treatment for 24 h in the
absence ( ) or presence (+) of 50 ng/ml TPA. Equal quantities of
protein were electrophoresed, transferred to nitrocellulose, and
immunoblotted with a C/EBP -specific antibody. As a positive control,
nuclear extract prepared from C/EBP expression vector-transfected
cells was electrophoresed in a parallel lane (lane C). The
arrow indicates the C/EBP protein, and the
numbers to the left of the panel show the
molecular mass of marker proteins (kDa). Similar results were observed
following an immunoblot of total cell extracts (not shown).
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The Role of AP1 and C/EBP Sites--
Previous studies indicate
that AP1 is an important mediator of TPA-dependent
regulation (20-22). Because the AP1-binding site is located adjacent
to the C/EBP-binding site (34), we wanted to assess the role of C/EBP
relative to AP1. We therefore evaluated whether both sites are required
for TPA- and C/EBP
-dependent activation of hINV
promoter activity. As shown in Fig. 7,
mutation of either site results in a loss of TPA- and
C/EBP
-dependent activation.

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Fig. 7.
C/EBP - and
TPA-dependent promoter activation requires the hINV
promoter C/EBP and AP1 sites. Keratinocytes were transfected with
2.5 µg of pINV-241, pINV-241(C/EBPm), or pINV-241(AP1-1m) luciferase
reporter plasmids in the presence of 50 ng of TPA/ml or 2.5 µg of
expression vector encoding C/EBP . After 24 h, the cells were
harvested, and extracts were prepared and assayed for luciferase
activity. The results are expressed relative to the activity observed
in non-C/EBP -transfected, non-TPA-treated cultures. The structures
of the mutated AP1 and C/EBP sites in pINV(AP1-1m) and pINV(C/EBPm) are
described under "Materials and Methods." The values are the average
of four separate determinations, and the bars represent the
standard error of the mean.
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DISCUSSION |
The hINV Promoter Proximal Regulatory Region--
The PRR of the
hINVpromoter is the region immediately upstream of the TATA box. This
region includes nucleotides
241 to
7, drives 50% of the activity
of the promoter, and contains the consensus binding site for
CCAAT/enhancer-binding protein AP1, the ets factors, and activator
protein-2 (14). Mutation of the C/EBP site results in the loss of
promoter activity. In contrast, mutation of the ets factor binding
sites (EBS-1, EBS-2) or the AP2-binding site changed basal promoter
activity but did not effect TPA
regulation.2 Our present
results suggest that the C/EBP site is essential for
TPA-dependent promoter activity. These results suggest a
dual role for C/EBP in maintaining basal promoter activity and in
mediating activation in response to TPA.
C/EBP Proteins Differentially Regulate hINV Promoter
Activity--
C/EBP proteins comprise a family of bZIP domain proteins
that readily form homo- and heterodimers (24, 25). These proteins are
important regulators of cell differentiation. For example, C/EBP
is
a transcriptional activator in adipocytes (35). High levels of C/EBP
and C/EBP
accumulate early in adipocyte differentiation but are
replaced by C/EBP
during late differentiation. C/EBP
and -
appear to play an important role in accelerating differentiation (24),
and ectopic expression of C/EBP
in multipotent NIH-3T3 cells causes
them to convert to committed adipoblasts. The accumulation of C/EBP
late in differentiation is correlated with expression of markers of
adipose differentiation (24, 36). C/EBP proteins also differentially
regulate gene expression in the liver (37, 38).
C/EBP proteins are also expressed in keratinocytes (39, 40). C/EBP
is a negative regulator of human papillomavirus transcription in
keratinocytes (39), and human C/EBP
is highly expressed in the
epidermis (40). Little is known regarding their role as regulators of
expression of keratinocyte genes; however, C/EBP
is expressed in
suprabasal epidermis and in differentiated keratinocytes (40). A recent
report indicates that C/EBP
, C/EBP
, and GADD153 are
differentially expressed during keratinocyte differentiation (41).
C/EBP
expression in the epidermis has not been studied. A recent
abstract suggests that C/EBP
directs expression of cytokeratin 10, a
keratin that is expressed in the suprabasal epidermal layers (42). Our
present experiments show that C/EBP proteins alter pINV-241 promoter
activity in a C/EBP site-dependent manner. C/EBP
increases basal transcription as efficiently as TPA treatment. In
contrast, C/EBP
and -
can suppress C/EBP
-dependent
activation. This is consistent with the ability of these proteins to
form zippered heterodimers and with the fact that different C/EBP
complexes differentially regulate gene expression (23-28). This result
shows that differential expression of C/EBP factors during keratinocyte differentiation could, in principle, differentially regulate involucrin gene expression.
TPA-dependent Increase in C/EBP
DNA Binding Is Not
Associated with an Increase in C/EBP
Concentration--
Our studies
indicate that the TPA-dependent activation of hINV promoter
activity is correlated with increased binding of C/EBP
to the hINV
promoter C/EBP site but that concentration of C/EBP
protein is not
altered by TPA treatment. Recent in vitro studies show that
C/EBP is a good substrate for protein kinase C but indicate that
protein kinase C-dependent phosphorylation reduces C/EBP
binding to DNA (43). This reduction in DNA binding is thought to result
from phosphorylation of serine residues within the DNA-binding region.
However, as discussed by the authors (43), this scenario has not been
confirmed in cells. It is possible that additional factors are
phosphorylated by protein kinase C that assist C/EBP
to bind DNA or
that, in vivo, serine phosphorylation actually enhances DNA
binding. Moreover, there may be cell type-specific effects. Additional
detailed studies will be necessary to determine how protein kinase C
alters C/EBP
phosphorylation in keratinocytes. However, it is clear
that the increased C/EBP
DNA binding is not the result of changes in
C/EBP
concentration.
GADD153 Suppresses C/EBP
- and TPA-dependent
Transcription--
GADD153 is a unique member of the C/EBP
transcription factor family that forms transcriptionally inactive
complexes with other C/EBP proteins. GADD153 forms heterodimeric
complexes with other C/EBP proteins that are unable to bind to DNA and
thus do not regulate transcription (27, 28). Thus, GADD153 can be used selectively to create C/EBP-null conditions (i.e. to
selectively knock out C/EBP function). We show that GADD153 inhibits
basal transcription. The fact that GADD153 suppresses basal promoter activity suggests that endogenous C/EBP proteins are involved in
maintaining basal promoter function. When C/EBP levels are artificially
elevated by transfection of C/EBP
, GADD153 also inhibits this
response, verifying the C/EBP protein requirement for promoter
activity. We also show that the TPA-dependent activation of
hINV promoter activity is suppressed by GADD153, suggesting that
TPA-dependent activation involves signal transduction
events that include C/EBP factors. This suggestion is supported by the finding that TPA treatment increases complex formation at the hINV
C/EBP-binding site and that C/EBP
is a part of this complex. C/EBP
is the primary C/EBP factor that interacts with the hINV C/EBP
site in cells maintained under basal conditions; no binding of C/EBP
or -
was detected. A possible mechanism to explain the increased
promoter activity in response to TPA is that TPA increases C/EBP
binding to the promoter. Consistent with this possibility, gel mobility
supershift experiments suggest that C/EBP
is an abundant component
of the C/EBP site binding activity present in TPA-treated cells.
Regulation of hINV Promoter Activity--
Involucrin gene
expression and promoter activity are regulated by a variety of
transcription factors. TPA is a potent inducer of keratinocyte
differentiation and an efficient activator of hINVgene expression and
promoter activity (20). TPA-dependent activation of
promoter activity has been shown to require the presence of functional
AP1 sites (20-22). The present study demonstrates the role of C/EBP
factors. These results are interesting for several reasons. First, the
C/EBP and AP1 sites in the hINV promoter PRR are separated by only 10 nucleotides (34). This close proximity should permit AP1-C/EBP factor
interaction. Second, both the AP1 and C/EBP-binding sites must be
intact for either TPA- or C/EBP
-dependent promoter
activation. This requirement suggests that C/EBP and AP1 factors
interact to produce the response and that members of both families are
required for activation. Understanding the mechanism of this regulation
will require further study. Third, promoter activity requires the
presence of both AP1 and C/EBP proteins (i.e. dominant
inhibitory proteins of either class inactivate the promoter) (44).
Fourth, both C/EBP and AP1 factor binding activity is increased by TPA
treatment (20). Taken together these features suggest that a
multiprotein complex, including C/EBP and AP1, may be required for hINV
gene expression. Our results suggest that this complex includes
C/EBP
.