From the
A rat homologue of chick ovalbumin upstream promoter
transcription factor I (COUP-TF I) was isolated using an expression
cloning method developed to iso-late neurite outgrowth inhibitors.
Overexpression of COUP-TF I in 3T3 fibroblasts resulted in reduction of
stable contact formation between neurites and transfected cells.
Additionally, COUP-TF I enhanced retinoic acid response
element-dependent reporter gene expression in 3T3 fibroblasts,
indicating that COUP-TF I can modulate transcriptional activation in
these cells. Our data suggest that COUP-TF transcription factors are
involved in the regulation of cell surface molecules during
neurogenesis.
Nuclear hormone receptors, which function as ligand-dependent
transcription factors, have a crucial role in establishing initial
cellular diversity in the nervous system. Among the best characterized
members of the superfamily of nuclear hormone receptors are the
receptors for retinoids, thyroid hormones, steroid hormones, and
glucocorticoids, whose importance during neurogenesis is well described
(for review, see Beato(1989); Evans and Arriza(1989); McEwen et
al.(1991); and Linney(1992)). Hormone receptors activate or
repress gene transcription through binding to cis-acting
hormone response elements (HRE)
Analyses of nuclear hormone
receptors in various systems clearly demonstrate that regulation of
gene expression by these transcription factors depends on the presence
of different ligands and also on the interactions with activators and
repressors. The role of COUP-TF orphan receptors in neurogenesis is
virtually unknown. In Drosophila, the expression of COUP-TF
homologue seven-up is required for development of specific
subset of photoreceptor neurons during eye development (Mlodzik et
al., 1990). In chick (Lutz et al., 1994) and mouse (Jonk et al., 1994; Lu et al., 1994; Qiu et al.,
1994), COUP-TFs are expressed in a complex spatial and temporal pattern
during development of the nervous system.
The central nervous system
of most adult vertebrates is inhibitory to axonal growth. During
development, inhibitory regions regulate the formation of neuronal
pathways and connections (Tosney and Landmesser, 1984; Davies et
al., 1990). Several factors responsible for neurite outgrowth
inhibition have been characterized (Caroni and Schwab, 1988; Cox et
al., 1990; Davies et al., 1990; Raper and Kapfhammer,
1990; Luo et al., 1993). We developed a screening system for
cloning inhibitory molecules after transfection of a cDNA library into
``substrate'' cells, which are subsequently cocultured with
neuronal cells and screened for functional expression of neurite
outgrowth inhibition. This approach allows isolation of cDNAs for
surface molecules, which cause direct inhibition of neurite outgrowth,
as well as cDNAs for factors involved in regulating the expression of
molecules, which inhibit or facilitate neurite outgrowth. This
screening system enabled us to isolate a rat homologue of orphan
receptor COUP-TF I, whose overexpression results in decreased contact
stability between neurites and substrate cells. These data suggest the
possible involvement of COUP-TF I in the regulation of neurite contacts
and cell aggregation.
To establish cell lines
overexpressing COUP-TF I, cDNA was cloned into the expression vector
pRcCMV (Invitrogen) using HindIII linkers and transfected into
3T3 cells followed by selection with G418 (600 µg/ml, Life
Technologies, Inc.) for 14-21 days.
Genomic DNA was prepared from isolated
clones (TurboGen, Invitrogen) and used as a template for amplification
of cDNAs by polymerase chain reaction. Primers for cDNA amplification
corresponding to flanking sequences in the pRcCMV vector (5`-primer
5`-AGCTCTCTGGCTAACTAGAGAAC, 3`-primer 5`-AGCGAGCTCTAGCATTTAGGTGA) were
prepared, and 35 cycles of polymerase chain reaction were performed
using the following conditions: 92 °C, 1.2 min; 58 °C, 2 min;
and 72 °C, 4 min. Amplified DNAs were cloned into EcoRV
site of Bluescript plasmid (Stratagene) for sequencing. Isolated cDNAs
with vector (pRcCMV) sequences were subcloned into pRcCMV expression
vector between HindIII and NotI sites and retested
for neurite inhibition.
For construction of reporter plasmids,
oligonucleotides containing corresponding HREs were synthesized,
trimerized, and cloned into the EcoRV site of Bluescript II KS
(Stratagene). Orientation of the oligonucleotides was determined by
sequencing. The Bluescript vectors containing oligonucleotides were
digested (HindIII/XbaI), and the fragments were
separated on a 10% polyacrylamide gel in TBE buffer, electroeluted, and
ligated to the HindIII/XbaI site of pBLCAT2 (Luckow
and Schütz, 1987).
The following oligonucleotides, based on
DR-1 (Kadowaki et al., 1992),
The role
of COUP-TF I as an activator or inhibitor of transcription in 3T3 cells
was examined by transient CAT assays using the
Members of the steroid hormone receptor superfamily interact
in a complex manner and stimulate or repress gene expression, depending
upon the type and number of factors present and also the context of the
gene promoter (Cooney et al., 1992, 1993). We have shown that
overexpression of the orphan receptor, COUP-TF I, can alter cell-cell
interaction in fibroblasts and block neuronal differentiation of PCC7
cells (Neuman et al., 1995). Expression of COUP-TF I in
fibroblasts is correlated with detectable alterations of normal cell
behaviors, presumably associated with alteration or modification of the
expression of cell surface proteins. The exact nature of this change is
unknown since no differences were detected in comparisons of isolated
membrane proteins from control and COUP-TF I-expressing cells, nor was
there any indication of involvement of secretory factors.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank
(
)(Green and
Chambon, 1988; Beato, 1989). Besides the ligand-dependent transcription
factors, the hormone receptor superfamily comprises several orphan
receptors for which ligands are not known (for review, see Evans(1988)
and Green and Chambon(1988)). Orphan receptors, chicken ovalbumin
upstream promoter transcription factor (COUP-TF) homologues, have been
isolated from Drosophila (Mlodzik et al., 1990), sea
urchin (Chan et al., 1992), zebrafish (Fjose et al.,
1993), Xenopus (Matharu and Sweeney, 1992), chick (Lutz et
al., 1994), and mammals (Miyajima et al., 1988; Wang et al., 1989; Richie et al., 1990; Wang et
al., 1991; Ladias and Karathanasis, 1991). One possible function
of COUP-TF transcription factors is to regulate the activity of
ligand-activated hormone receptors through heterodimer formation or
competition for specific response elements (Cooney et al.,
1992, 1993; Tran et al., 1992).
Cell Culture
Mouse NIH-3T3 fibroblasts
and neuroblastoma glioma hybrid NG108-15 (Hamprecht,
1977) cells were grown in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum (Sigma). Neuronal differentiation of NG108-15 cells was
achieved by culture in serum-free Dulbecco's modified
Eagle's medium for 72 h.
Generation of Subtraction cDNA
Library
Poly(A) RNA was isolated
(FastTrack, Invitrogen) from brains and spinal cords of embryonic day
17, postnatal day 5, and adult rats and also from chronically injured
spinal cords. 24 µg of Poly(A)
RNA from postnatal
day 5, adult, and injured spinal cord (8 µg from each) was used to
synthesize first strand cDNA using oligo(dT) primer with a NotI restriction site at the 5`-end
(5`-CTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTTT) andSuperScript
RNaseH-reverse transcriptase (200 units/µg of Poly(A)
RNA, Life Technologies, Inc.). The first strand synthesis
reaction was labeled using [
P]dCTP (5 µCi,
>3000 Ci/mmol, Amersham Corp.) to facilitate tracing of the DNA.
First strand cDNA was hybridized to embryonic day 17 poly(A)
RNA (200 µg) in sealed ampules (total volume, 100 µl,
buffer 0.5 M sodium phosphate, pH 6.8, 300 mM NaCl, 2
mM EDTA, and 0.2% SDS) for 18 h at 70 °C. The
hybridization mix was diluted to a final molarity of 0.08 M sodium phosphate, loaded onto a DNA grade hydroxylapatite column
(4 ml (volume), Bio-Rad), and washed extensively with 0.08 M sodium phosphate buffer (pH 6.8). Single-stranded cDNAs were
eluted in 10 ml of 0.15 M sodium phosphate buffer. Column
fractions (0.5 ml) exhibiting radioactivity above background were
pooled, and the cDNAs were concentrated by butanol extraction followed
by chromatography in STE buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) on a Sephadex G-25 column
(Pharmacia Biotech Inc.). Single-stranded cDNAs were mixed with 100
µg of poly(A)
RNA from embryonic day 17 for the
second cycle of hybridization. After the second cycle, first strand
cDNAs were hybridized with 5 µg of the original Poly(A)
RNA isolated from postnatal day 5, adult, and injured rats
nervous systems, and the resulting DNA/RNA hybrids were used as
template for second strand synthesis with RNaseH and Escherichia
coli DNA polymerase I (Life Technologies, Inc.). Blunt ends were
created with T4 DNA polymerase, and the HindIII adapter
(5`-AGCTTGGCACGAG-3`, 3`-ACCGTGCTC-5`) was ligated to the cDNA. Longer
cDNAs (>700 bp) were isolated by digestion with NotI
followed by size selection on a Sephacryl S 400 column (Pharmacia) and
were subsequently cloned between the HindIII and NotI
restriction sites of the expression vector pRcCMV (Invitrogen). The
library was divided into 20 aliquots and used to transform E. coli DH5
cells (MAX Efficiency, Life Technologies, Inc.). Each
aliquot yielded 5-8
10
colonies, which were
combined and grown for large scale plasmid isolation (plasmid maxi kit,
Qiagen). The cDNA expression library has 2
10
independent clones with an average insert size of 1.5 kilobases
(range, 0.6-3.7 kilobases).
Isolation of Cell Clones and
cDNA
Transfection of the cDNA library into fibroblasts was
performed by the calcium phosphate coprecipitation technique using 20
µg of DNA/100-mm tissue culture plates (Falcon) at a cell density
of 2 10
cells per plate with an incubation time of
15-16 h. Each aliquot of the cDNA library (n =
20) was used to transfect cells in 20 plates. The cDNA library was
transfected into NIH-3T3 fibroblast cells. Selection of transfected
cells was performed with 600 µg/ml G418 (Life Technologies, Inc.)
for 14-21 days prior to testing for neurite outgrowth inhibition.
Screening for neurite outgrowth inhibition involved coculture of
transfected fibroblasts with the neuroblastoma-glioma hybrid cell line
NG108-15 differentiated for 72 h in serum-free culture medium
prior to coculture. Differentiated NG108-15 cells rapidly
established an extensive and robust network of contacts with control
NIH-3T3 cells, indicating that fibroblasts provide a good substrate for
contact formation. Inhibitory clones were identified as those that had
fewer or no contacts with surrounding neurites. Inhibitory clones were
isolated and propagated.
Northern Blot Analyses
Total RNA was
isolated using acid guanidinium/phenol/chloroform extraction procedure
(Chomczynski and Sacchi, 1987). The RNA (25 µg/lane) was
fractionated on 1.2% agarose-formaldehyde gel and transferred to a
nylon membrane (Hybond N, Amersham). Full-length COUP-TF I was
radiolabeled (P) using Multiprime DNA labeling system
(Amersham) and used as a probe. The blots were washed at high
stringency (0.2
SSC, 65 °C) and exposed to x-ray film for 3
days. Transferred RNAs were monitored by methylene blue staining of the
filters before hybridization.
Analyses of Contact Stability between Transfected
Fibroblasts and Neurites of NG108-15 Cells
Fibroblasts
were plated in 12-well dishes at a density of 2 10
cells/well 72 h prior to coculture. Differentiated NG108-15
cells were added at a density of 2
10
cells per
well. 2 h after coculture initiation, fibroblast colonies were
identified, and the number of contacts between fibroblasts and
NG108-15 cells were recorded at three time points: 1) starting
time, t = 0; 2) 0.5 h later, t = 0.5;
and 3) 2.5 h after the starting time, t = 2.5. The
number of contacts was used to determine the direction and magnitude of
any change in the dynamics of cell-cell interaction. Analysis of the
data was performed with one-tailed t test.
Transfections and CAT Assays
Cells (3
10
) in 60-mm dishes were cotransfected overnight
with a combination of plasmid DNA (total 15 µg) by the calcium
phosphate coprecipitation method. The DNA mix consisted of expression
plasmid (pRcCMV or pRc-COUP-TF I, 9 µg), CAT reporter gene
construct (DR-1,
-RARE, CRBP I, 3 µg), and pRcCMV-lacZ (3
µg). The pRcCMV-lacZ construct was used to facilitate normalization
of the CAT activity to transfection efficiency. Transfections were
performed in triplicate. Transfection medium was replaced after 18 h
with a control media or media supplemented with 10
M retinoic acid. Cells were harvested 48 h later and
processed for CAT and lacZ assays according to the methods of Sambrook et al. (1989). Protein concentration was determined with a
protein assay reagent (Bio-Rad) using bovine serum albumin as a
standard. Samples from the CAT assays were resolved by thin-layer
chromatography, visualized on x-ray film, and quantitated by liquid
scintillation counting.
-RARE (Tran et
al., 1992), and CRBP I (Tran et al., 1992), were
synthesized: DR-1, 5`- GGCTTCAGGTCAGAGGTCAGAGA and 5`-
GGTCTCTGACCTCTGACCTGAAG;
-RARE, 5`-GGTGTAGGGTTCACCGAAAGTTCACTCA
and 5`-GGTGAGTGAACTTTCGGTGAACCCTACA; and CRBP I,
5`-CCATCCAGGTCAAAAAGTCAGGA and 5`-GGTCCTGACTTTTTGACCTGGAT. The HRE
sequences are underlined.
Preparation of Whole Cell Extracts and
Electrophoretic Mobility Shift Assay (EMSA)
These methods
were performed at 4 °C as previously described (Scholer et
al., 1989). Briefly, the extraction buffer contained 20
mM Hepes, pH 7.8, 450 mM NaCl, 0.2 mM EDTA,
0.5 mM dithiothreitol, 25% glycerol, and protease inhibitors
phenylmethylsulfonyl fluoride (0.5 mM), leupeptin (0.5
µg/ml), pepstatin (0.7 µg/ml), aprotinin (1 µg/ml), and
bestatin (40 µg/ml). Sonicated extracts were cleared by
centrifugation for 5 min. Binding conditions for the EMSA were 10
mM Hepes, pH 7.8, 1 mM spermidine, 5 mM MgCl, 50 mM KCl, 0.5 mM
dithiothreitol, 9% glycerol, 0.8 µg of poly(dI-dC), 50,000 cpm of
the
P-labeled oligonucleotide, and 10 µg of cell
extract. After incubation for 15 min at 25 °C, DNA-protein
complexes were separated on a 5% polyacrylamide gel, followed by
autoradiography. Three different batches of extracts were used in EMSA
analyses, and no batch differences were observed.
Cloning of COUP-TF I
Expression
screening of the subtraction cDNA library was performed to isolate
genes that inhibit neurite outgrowth. The cDNA library in pRcCMV
expression vector was transfected into NIH-3T3 fibroblasts, and, after
3 weeks of antibiotic selection, several clones that had less contacts
with neurites were isolated. These clones were propagated, and the
transfected cDNAs were isolated using polymerase chain reaction and
retested for neurite outgrowth inhibition. Sequence analysis revealed
that one cDNA is the rat homologue of human orphan receptor COUP-TF I
lacking 40 amino acids of the transactivation domain localized near the
amino terminus of the DNA binding domain (COUP-TF I). Full-length
COUP-TF I cDNA was isolated by high stringency screening of the brain
cDNA library cloned into
ZAPII vector (Stratagene). The rCOUP-TF I
cDNA encodes a protein with a predicted molecular mass of 45.8
kilodaltons (Genbank accession number, U10995) and is 98% identical to
the human COUP-TF I at the amino acid level with 95% identity at the
nucleotide level.
COUP-TF I Interacts with HREs in
Fibroblasts
Expression of COUP-TF I was analyzed by
Northern blot using six COUP-TF I and three pRcCMV vector-transfected
clones. Vector-transfected and -untransfected fibroblasts did not
express detectable levels of COUP-TF I (Fig. 1A, lanes1, 9, and 10). Three
transfected clones expressed introduced COUP-TF I at high levels (Fig. 1A, lanes6, 7, and 9), two clones expressed at moderate levels (Fig. 1A, lanes3 and 5),
while one clone did not express COUP-TF I at a detectable level (Fig. 1A, lane4).
Figure 1:
Expression and
activity of COUP-TF I in transfected fibroblasts. A, Northern
blot analyses of COUP-TF I expression in control 3T3 fibroblasts (lane1) and fibroblast clones transfected with
COUP-TF I cDNA cloned into pRcCMV expression vector (lanes2-8) or pRcCMV vector only (lanes9 and 10). Expression of COUP-TF I was high in clones
7-9 (lanes6-8, respectively) and varied
from undetectable to moderate in clones 2-5 (lanes2-5, respectively). B, EMSA analysis of
control fibroblasts (lane 1) and fibroblasts transfected with
pRcCMV expression vector (lane2) and COUP-TF I cDNA
in pRcCMV (lane3). Cell extracts were analyzed by
EMSA using oligonucleotides DR-1, -RARE, and CRBP I. As a control
for binding specificity, 100 times molar excess of corresponding
unlabeled oligonucleotides were added to the binding reaction (lane4). C, the transient CAT activity of DR-1-TK-CAT (DR-1),
-RARE-TK-CAT (
-RARE), and CRBP
I-TK-CAT (CRBP) reporter constructs in the presence of pRcCMV (CMV) or COUP-TF I-expressing plasmid pRcCMV-COUP-TF I (COUP). Subsets of each transfection group were treated with
10
M retinoic acid (RA). All cells
were cotransfected with pRcCMV/lacZ constructs to facilitate
normalization of CAT gene expression to transfection efficiency. All
treatments were done in triplicate, and the numbers represent
values of two experiments. Values are normalized based upon
transfection efficiency and are expressed as relative to the results
obtained by transfection of pRcCMV-transfected cells with corresponding
reporter plasmid for which activities were set at 1. Results were
analyzed by a one-tailed t test.
Since COUP-TF I
can form inactive complexes with retinoid X receptors (RXR) and binds
to HREs from different genes as a homodimer to repress the hormone
response (Cooney et al., 1992, 1993; Tran et al.,
1992), we used EMSA analysis to examine whether the overexpression of
COUP-TF I in fibroblasts affects formation of HRE binding complexes.
Since the effects of COUP-TF I depend on the HRE sequence, we analyzed
two natural and one synthetic HRE: -RARE, a direct repeat with a
5-bp spacer that is activated by RAR
and is not inhibited by
COUP-TF I (Tran et al., 1992); CRBP I-RARE, a direct repeat
with a 2-bp spacer that is optimally activated by RAR
RXR
heterodimers but not by RXR homodimers (Hermann et al., 1992;
Zhang et al., 1992); and DR-1, a direct repeat that contains a
1-bp spacer and has a high affinity for COUP-TF I (Kadowaki et
al., 1992) and RXRs (Mangelsdorf et al., 1990, 1991). Our
results demonstrated that the pattern of protein-DNA complexes is
changed in COUP-TF I overexpressing cells compared with controls (Fig. 1B). A unique DNA-protein complex (Fig. 1B, arrow) was detected in extracts made
from COUP-TF I overexpressing cells. Antibodies (developed in our
laboratory) against COUP-TF I supershifted the complex (data not
shown), verifying the presence of COUP-TF I in the complex.
-RARE, CRBP I, and
DR-1 oligonucleotides coupled to the TK promoter of the CAT reporter
gene. Results from cotransfections of reporter constructs and pRcCMV or
COUP-TF I expression vectors are shown in Fig. 1C.
Cotransfection of COUP-TF I significantly induced (p <
0.05)
-RARE containing reporter construct activity (Fig. 1C). Addition of retinoic acid-induced expression (p < 0.01) of the
-RARE reporter gene construct in
cells cotransfected with the pRcCMV control expression vector to levels
similar (p > 0.05) to cells cotransfected with COUP-TF I in
the presence or absence of retinoic acid. No significant effects of
COUP-TF I or retinoic acid treatment on reporter gene expression of
other constructs was detected. In these experiments, COUP-TF I
functioned as a positive regulator of
-RARE-coupled reporter gene
expression in 3T3 fibroblasts.
COUP-TF I Overexpression Changes Contact Stability
between Transfected Fibroblasts and Neurites
The growth
rate of neurites from NG108-15 cells is rapid and robust, which
makes them a good model for studying cell-cell interaction and contact
stability. Analyses of neurite contact stability were performed with
fibroblast clones overexpressing COUP-TF I. Fibroblasts transfected
with the pRcCMV vector only and naive fibroblasts were used as
controls. There is a general increase in the number of contacts over
the course of the experimental period. This increase was not different
for naive control fibroblasts or fibroblasts transfected with the
expression vector (205 ± 64% versus 160 ± 37%,
respectively, p > 0.05). However, fibroblasts transfected
with COUP-TF I (clones 5, 8, and 9) exhibited a significantly (p < 0.05) lower increase in contact number (51 ± 14%, 18
± 20%, 38 ± 27%, respectively, Fig. 2) indicative
of lower contact stability. While differences in level of expression of
COUP-TF I were apparent in clones 5, 8, and 9 (Fig. 1A, lanes5, 7, and 9, respectively),
contact stability was not dependent upon the level of expression of
COUP-TF I cDNA (p > 0.05). Neuroblastoma-glioma
NG108-15 cells have rapidly growing neurites with large growth
cones and filopodia. After contact with control fibroblasts, growth
cones maintain their form (Fig. 3, CMVpanel).
In contrast, the processes of NG108-15 cells exhibit retraction
or become very small after contact with COUP-TF I-expressing cells,
indicative of growth cone collapse and contact instability (Fig. 3, COUPpanel). The increased number of
contacts in these cultures comes from other NG108-15 cells in the
vicinity of the fibroblasts. Since the differentiated NG108-15
cells observed have an equal probability to establish and maintain
contacts with fibroblasts, the test determines the stability of
cell-cell contact.
Figure 2:
Cells expressing COUP-TF I exhibit lower (p < 0.05) contact stability when cocultured with
NG108-15 cells. Contact stability was assayed in three groups: 1)
naive fibroblasts (control); 2) cells transfected only with pRcCMV
vector (CMV 3); and 3) three clones transfected with COUP-TF I cDNA
(COUP 5, COUP 8, and COUP 9). The number of contacts between cell types
at time 0 and 2.5 h was used to calculate the percentage change in the
number of contacts for each colony of cells observed. The bars represent the average percentage change in the number of contacts
observed for each group and represent the data from three separate
experiments. The number of observations (n) for each group is
given.
Figure 3:
Interactions of neurites with control and
COUP-TF I overexpressing 3T3 fibroblasts. 2 h after the addition of
NG108-15 cells (t = 0), fibroblast clones were
identified and photographed at various times during the culture period
(given below each panel). Fibroblasts transfected
with the pRcCMV control vector (F in upperCMVpanel) provide a good substrate for support of processes
from NG108-15 cells (N). The processes are robust, and
contacts (arrows) are persistent during prolonged culture. In
contrast, cells overexpressing COUP-TF I cDNA (F in lowerCOUPpanel) do not sustain significant
attachment of processes from NG108-15 cells. During the culture
period, contacts (arrows) are not
maintained.
(
)Discerning the mechanism by which COUP-TF I altered
surface properties was further hampered due to a lack of
characterization of fibroblasts with regard to members of the steroid
hormone receptor superfamily, which are endogenously expressed and
represent potential heterodimerization partners. However, data from
transient CAT assays indicated that COUP-TF I induced transcriptional
activity of
-RARE-coupled reporter gene constructs. Together,
these data are consistent with a model in which COUP-TF I interacts
with other nuclear hormone receptors, causing changes in gene
expression. Several scenarios are possible, including COUP-TF I
homodimers binding to retinoic acid response elements, or COUP-TF
I/retinoic acid receptor heterodimer formation (Tran et al.,
1992) with subsequent alteration of gene expression. Retinoic acid
alters expression of genes that promote cell growth and attachment, i.e. laminin B1 (Vasios et al., 1991), and is also
important in the regulation of hox genes (Linney, 1992), which
have been shown to interact with promoters of cell adhesion molecules, i.e. N-CAM and cytotactin (Jones et al., 1992a,
1992b, 1993). Our data are consistent with a model in which
perturbation of a variety of processes, including differentiation,
aggregation, and cell-cell interaction, is regulated by expression of
COUP-TF I.
/EMBL Data Bank with accession number(s)
U10995.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.