(Received for publication, October 17, 1995)
From the
The temporal expression of the early growth response gene (EGR-1) is one molecular mechanism for both maximal activation
of the G gene and accelerated growth in mitotically
active predifferentiated LLC-PK
renal cells. These events
are dependent on an enhancer area in the 5`-flanking region of the
G
gene that contains an EGR-1 motif
(5`-CGCCCCCGC-3`). However, acquisition of the polarized phenotype in
LLC-PK
cells is accompanied by loss of EGR-1 expression and occupancy of the EGR-1 site by nuclear
binding proteins other than EGR-1. We now demonstrate that one of these
binding proteins is the Wilms' tumor suppressor (WT1).
Furthermore, the temporal expression of WT1 in LLC-PK
cells acquiring the polarized phenotype represses both
G
gene activation and growth in these cells. These
findings suggest the existence of differentiation-induced pathways in
LLC-PK
cells that alternatively abrogrates EGR-1 and promotes WT1 gene expression, thereby modulating a
target protooncogene G
that is participatory for
growth and differentiation in renal cells. These studies emphasize the
usefulness of the LLC-PK
renal cell as a model to elucidate
normal programs of genetic differentiation in which WT1 participates.
Wilms' tumor (WT) ()is a pediatric nephric
neoplasm arising from the continued proliferation of embryonic
blastemal cells that fail to differentiate(1) . WT occurs in
both sporadic and hereditary forms and as part of the WAGR syndrome
(Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation)(2) . Chromosome 11p13 contains a region encoding
the tumor suppressor gene WT1, which is commonly deleted in
heritable WT(3, 4) . Internal deletions and mutations
of the WT1 gene are found in some WTs (reviewed in (5) ). Although WT1 is expressed in uterus, spinal cord,
spleen, abdominal wall musculature, and the mesothelial lining of
organs within the thoracic cavity, its highest expression is in the
developing urogenital system(6) . The WT1 gene encodes
four splice variants of a transcription factor containing a
glutamine/proline-rich N terminus and four carboxyl
Cys
His
type zinc fingers, which recognize
5`-GCGGGGGCGGTG-3` and redundant 5`-TCC-3` cis motifs (7, 8, 9) . Alternative splice I allows the
variable insertion of exon 5 between the transactivating and DNA
binding domains of WT1. Alternative splice II allows the variable
insertion of nine nucleotides encoding lysine, threonine, and serine
(+KTS or -KTS) between the third and fourth zinc
fingers(7) . WT1 cis motifs are also recognized by
another Cys
His
type zinc finger transcription
factor EGR-1 also known as NGF1-A, Krox 24, TIS-8, and Zif 268, which
is induced after mitogenic and differentiation cues in several cell
types (reviewed in (10) ). In contrast to EGR-1, WT1 appears to
predominantly function as a transcriptional repressor of several
growth-related genes including EGR-1, insulin-like growth factor II,
platelet-derived growth factor
chain, insulin-like growth factor
I receptor, colony-stimulating factor 1, transforming growth factor
1, and Pax2 (reviewed in (11) ). WT1 mutations
prevent DNA binding or transcriptional repressive functions that are
postulated to allow the constitutive expression of growth factors
leading to renal neoplasia. Consistent with this possibility,
suppression of colony formation occurs in WT cells transfected with
wild type WT1 isoforms(12) .
Further insights into molecular
cascades linking WT1 to target genes in renal cells would clearly be
advantageous. WTs occur with greater frequency in pediatric kidneys
containing persistent renal stem cells, a condition designated
nephroblastomatosis or nephrogenic rests. WT1 mutations occur
in these rests, suggesting they may represent a transitional cell
preceding malignancy(13) . Embryonal kidney cell tumors
reminiscent of WT can be induced in rats given the alkylating agent N-nitroso-N`-methyl urea(14) . However,
cultured cells have yet to be developed from these sources. We have
previously identified genes participatory for epithelial cell growth
and differentiation events in LLC-PK cells, an extensively
characterized cultured epithelial cell line derived from juvenile male
pig kidney(15) . These studies indicated that as in lower
eukaryotic organisms such as Dictyostelium(16, 17, 18) and Drosophila(19) , heterotrimeric guanine nucleotide
binding (G) proteins are involved in signal transduction pathways
required for both growth and cellular differentiation programs in renal
cells.
G proteins are composed of individual ,
, and
subunits that are encoded by gene superfamilies that have been
conserved by eukaryotes throughout evolution (reviewed in (20) ). Most of the transducing activities of G proteins in
mammalian cells are associated with the state of activation of the
subunit, which is involved in GDP/GTP exchange and GTP hydrolysis
(reviewed in (21) and (22) ). G proteins can alter
cell growth or differentiation by participation in growth factor
receptor signaling pathways that converge in the nucleus to alter gene
expression. Mutations in G
comparable with Ha-ras
GP21, which decrease GTPase activity, are found in tumors of the
adrenal cortex and ovary(23) . Such mutations, which convert
the G
gene into the oncogene gip2 induce increased
growth and oncogenic transformation in Rat-1a cells(24) .
Increased growth may be a consequence of persistent activation of
pathways coupled to mitogen-activated protein kinase (25) . The
G
subunit interacts with pathways required for
differentiation of F9 teratocarcinoma cells(26) . Even modest
repression of G
expression is associated with renal
developmental and morphologic abnormalities in transgenic mice,
underscoring its important role in renal differentiation
events(27) . Polarized LLC-PK
cells contain two
G
isoforms, G
and
G
, which are involved, respectively, in the
regulation of hormone-stimulated adenylyl cyclase and constitutive
proteoglycan secretion through the Golgi
complex(28, 29) . The genes encoding both G
subunits are transcriptionally activated in these cells in a
coordinated manner during growth and differentiation but differ in
response to glucocorticoids and
cAMP(30, 31, 32) . We recently determined in
mitotically active predifferentiated LLC-PK
cells that the
temporal expression of EGR-1 is one molecular mechanism for
both maximal activation of the G
gene and
accelerated growth in these cells. These events were dependent on an
enhancer area in the 5`-flanking region of the G
gene that contains an EGR-1 motif
(5`-CGCCCCCGC-3`)(33) . Notably, acquisition of the polarized
phenotype in LLC-PK
cells was accompanied by loss of EGR-1 expression and occupancy of the EGR-1 site by
nuclear binding proteins other than EGR-1. In the present study we
determine whether one of these binding proteins is WT1.
LLC-PK cells plated on
glass coverslips were fixed for immunofluorescent staining on Days
1-7. Cells were fixed in 4% paraformaldehyde for 1 h,
permeabilized in Triton X-100 for 4 min, and then incubated in PBS
containing 0.1% bovine serum albumin for 5 min to reduce nonspecific
background staining. The cells were incubated for 2 h in anti-EGR-1 Wi
21 (alpha 1), anti-WT1
6F or H7, preimmune rabbit IgG, or
non-immune murine ascites at 1:50 or 1:100 dilutions, washed three
times in 0.1% bovine serum albumin in PBS, and then incubated for 1 h
with goat anti-rabbit or anti-murine IgG conjugated to fluorescein
isothiocyanate (Kirkegaard and Perry). Cells were washed three times in
PBS, then mounted in 100 mM Tris-HCl:glycerol, 50:50, 2% n-propyl gallate, pH 8, and viewed on an Olympus BHS
photomicroscope equipped for epifluorescence.
During culture, LLC-PK cells differentiate from a
rounded cell type to a fully polarized epithelium. Prior to their
polarization and tight junction formation, these cells undergo several
rounds of cell division coincident with the maximal EGR-1 expression and activation of the G
gene(31) . To determine whether the WT1 gene also
participates in these events a full-length human WT1 cDNA and
a polyclonal (
6F) or monoclonal (H7) antibody to the 173-residue
amino-terminal non-zinc finger region of the WT1 protein were used to
detect its expression in LLC-PK
cells. As seen in Fig. 1C, immunofluorescence of dividing non-confluent
non-polarized cells with this antibody revealed a bright nuclear
staining pattern in virtually every cell for EGR-1 whereas WT1 was not
detected. By contrast, in fully polarized confluent cells this staining
pattern was reversed. Quantification of EGR-1 and WT1 proteins in cells
at Days 1-7 after plating by immunoblotting revealed a reciprocal
pattern of expression for each protein in these cells. EGR-1 was barely
detectable immediately after plating whereas WT1 was well expressed.
However, by 36-48 h, when the cells were actively dividing, EGR-1
protein was maximally expressed whereas WT1 was not detectable. On
later culture days 5-7, following the full polarization of these
cells, EGR-1 expression fell whereas WT1 protein and mRNA
expression increased (Fig. 1, A and B). In day
7 cultures there was sporadic lifting of some quiescent cell monolayers
(data not shown). Ingrowth of cells occurred in the margins of denuded
areas of the culture plates with a corresponding re-expression of EGR-1
protein. The pattern of maximal expression of the EGR-1 protein
coincides with the temporal maximal activation of the
G
gene during growth, whereas its repression
coincided with the maximal expression of the WT1 protein during cell
quiescence(31) .
Figure 1:
Detection of WT1 gene
expression in cultured LLC-PK cells. A, Northern
blot analysis of a tRNA(-), Wilms' tumor (+), and
LLC-PK
RNA from culture days 0 (day of trypsinization)
through day 7 with a full-length human WT1 cDNA detected a
predominant
3-kilobase transcript. B, quiescent
LLC-PK
cells from day 10 of culture were trypsinized (day
0) and cultured for successive days. Immunoblotting of total cell
extract from each of these cultures was performed to compare the
relative amounts of EGR-1 or WT1 protein present. Each lane represents 150 µg of protein quantitated. Autoradiograms of
these proteins from the same acrylamide gel show in the upper panel EGR-1 induction (
80 kDa) on culture day 1. The lower
panel II shows maximal induction of WT1 protein (
50 kDa) on
culture days 5-7. Immunoprecipitation of WT1 was concurrently
performed in these cultures, which also showed maximal induction of WT1
protein on culture days 5-7 seen in panel I. C,
cell monolayers at days 1-10 were fixed, permeabilized, and
stained by immunofluorescence with immune rabbit IgG anti-EGR-1 Wi 21
(alpha 1) (which detects the non-zinc finger region of the EGR-1
protein corresponding to residues 29-117) or
6F and H7
antibodies (which detect the non-zinc finger amino-terminal region of
the WT1 protein corresponding to residues 1-173). Representative
images of cell cultures at days 2 (left panel) and 7 (right panel) are shown. No staining was observed with
preimmune rabbit IgG or non-immune mouse ascites (data not
shown).
In other cell types the overexpression of
the G subunit can activate signaling pathways that
contribute to accelerated cell growth. Likewise overexpression of
G
or EGR-1 also accelerates LLC-PK
cell growth. As WT1 expression corresponded to culture
times when LLC-PK
cells were both differentiated and growth
arrested, we questioned whether expression of WT1 in predifferentiated
LLC-PK
cells would antagonize the effects of EGR-1 on cell
growth during this period. LLC-PK
cells were transiently
transfected with Bluescript or plasmids encoding cDNAs for
G
(pRSV G
) or WT1 (pRSV
WT1), driven by a viral Rous sarcoma promoter-enhancer to overexpress
each protein. As seen in Fig. 2, by day 3 of culture
predifferentiated cells transfected with plasmids encoding
G
had growth rates that were 1.8 times faster than
cells transfected with Bluescript, whereas LLC-PK
cells
transfected with WT1 had growth rates that were 35% lower than
Bluescript-transfected cells. Transfection efficiency in all these
experiments was comparable at approximately 10-20% efficiency.
Despite the initial altered growth rates of G
and WT1-transfected cells, they still developed a normal polarized
phenotype upon achieving confluence. These data suggested that both
G
and WT1 participate in signaling pathways
that normally contribute to renal cell growth.
Figure 2:
Growth curves of LLC-PK cells
in 10% fetal calf serum. Squares depict cells transfected with
plasmids encoding G
, triangles depict cells
transfected with plasmids encoding WT1, and circles depict cells transfected with Bluescript II KS. Results are
expressed as the mean cell number ± S.E. of 12 independent
observations.
We previously
documented in rapidly dividing non-polarized cells a 135-bp enhancer
area in the -200 to -335 region of the G gene. This region contains a binding site (5`-CGCCCCCGC-3`) for
the EGR-1 transcription factor that provides a genomic signaling
pathway for mitogenesis(33) . To assess whether WT1 was
repressing cell growth by repressing maximal transcriptional activation
of the G
gene, cells were co-transfected with the
pRSV WT1 plasmid, and plasmids encoding firefly luciferase
reporter genes fused to 5`-flanking areas of the G
gene with (M14) or without the EGR-1 site (mutated M14
and M4). As seen in Fig. 3, a 60% repression of G
transcription was only found in renal cells following their
transfection with the M14 plasmid that contained both an intact EGR-1 binding site and also overexpressed a functional WT1
protein. These data suggested that the WT1 protein was contributing to
the temporal transcriptional repression of the G
gene in LLC-PK
cells during culture.
Figure 3:
Transcription of G genes in polarized LLC-PK
cells. Upper
panel, plasmids M14 (black bars), mutated M14 (gray
bars), and M4 (striped bars), which represent serial
deletions of 10-4 LUC-(XmaIII) fused to a firefly
luciferase cDNA, were co-transfected with Bluescript II KS or a plasmid
encoding WT1, pRSV WT1. These cells were then
examined for luciferase activity. Luciferase activity results are
expressed as percent of control in light units ± S.E. of
constructs transfected with WT1 above those transfected with
Bluescript II KS, normalized for human placental alkaline phosphatase
activity in each of six individual deletion experiments (100% indicates
unity or no change from control). Lower panel, sequence of the
G
gene indicating the respective start sites for
deletions of M14 and M4 plasmids. The EGR-1 consensus sequence
``CGCCCCCCGC'' is underlined. Sites of the created
mutation in M14 are shown above.
To determine
whether the WT1 protein was directly contributing to the activation of
the G gene, a double-stranded 23-bp DNA segment
derived from the 5`-flanking sequence of the gene, which also contained
the EGR-1 consensus sequence (5`-ATCCGCC CGCCCCCGCCGTCGGG-3`),
was synthesized and
P end-labeled for direct binding
studies in mobility shift assays. Nuclear extracts from LLC-PK
cells that were either actively dividing and non-polarized
(culture day 1) or LLC-PK
cells that were relatively
quiescent and fully polarized (culture day 7) were examined. As seen in Fig. 4, the binding patterns of nuclear extracts from culture
day 1 were different from those on culture day 7. Nuclear extracts from
day 7 cells consistently demonstrated an additional faster mobility
complex. We previously identified the EGR-1 protein as a predominant
component of nuclear binding proteins in day 1 dividing cells but not
day 7 quiescent cells (33) . Based on our immunocytochemical
studies it would be anticipated that the WT1 protein should be present
in nuclear extracts of quiescent fully polarized LLC-PK
cells on culture day 7. To determine whether WT1 was one of the
proteins interacting with the 23-bp probe, nuclear extracts from
culture days 1 and 7 were preincubated with H7 antibody. Following
electrophoresis, retarded mobility of only the additional complex in
nuclear extracts from culture day 7 was found. The specificity of this
interaction was demonstrated by competition with the 28-kDa protein
expressing the amino-terminal 173 residues of the WT1 protein. These
data demonstrate that the WT1 protein was one component of these
nuclear complexes. Detectability of the WT1 protein was consistent with
its pattern of maximal expression in quiescent polarized
LLC-PK
cells that also have a corresponding repression of
the G
gene.
Figure 4:
Mobility shift assays with the 23-bp DNA
segment of the G gene containing the EGR-1 binding site identify WT1 as a component of an induced nuclear
complex in quiescent polarized LLC-PK
cells. Nuclear
extracts were prepared from LLC-PK
cells 24 or 144 h after
trysinization and culture. A 23-bp DNA segment containing the EGR-1 consensus sequence (5`-ATCCGCCCGCCCCCGCCGTCGGG-3`) was used as a
probe in mobility shift assays. Lane 1, probe alone; lane
2, probe with 24-h nuclear extracts; lane 3, probe with
24-h nuclear extracts preincubated with non-immune ascites; lane
4, probe with 24-h nuclear extracts preincubated with immune
ascites
6F; lane 5, probe with 144-h nuclear extracts; lane 6, probe with 144-h nuclear extracts preincubated with
non-immune ascites; lane 7, probe with 144-h nuclear extracts
preincubated with immune ascites H7; lane 8, probe with 144-h
nuclear extracts preincubated with immune ascites H7 and albumin; lane 9, probe with 144-h nuclear extracts preincubated with
immune ascites H7 and competed with recombinant WT1 protein to which H7
was generated. Note similar data were obtained with the
6F
antibody. The arrow indicates the position of the faster
mobility complex detected in nuclear extracts 144 h after
trypsinization and culture.
These findings suggest the existence
of differentiation-induced pathways in LLC-PK cells that
alternatively abrogrates EGR-1 and promotes WT1 gene
expression, thereby modulating a target protooncogene G
that is participatory for growth and differentiation in these
renal cells. These studies emphasize the usefulness of the LLC-PK
renal cell as a model to elucidate normal programs of genetic
differentiation in which WT1 participates. Further examination
these pathways may provide significant insights into the molecular
events involved in renal hypertrophy, nephrogenesis, and oncogenesis.