(Received for publication, April 12, 1994; and in revised form, November 7, 1994)
From the e 10, 13125 Berlin, Germany
and ICRF, Cell Interaction Laboratory, Cambridge University
Medical School, Cambridge CB2 2QH, United Kingdom
Scatter factor/hepatocyte growth factor (SF/HGF) and its
receptor c-Met represent a paracrine signaling system involved in
mesenchymal-epithelial interactions during development and during tumor
progression. We have examined the promoters of the mouse and human
SF/HGF genes by deletion mapping followed by CAT assays as well as by
gel retardation and footprinting analysis. The promoter sequences are
highly conserved (89.5% identity) up to position -453 from the
major transcription start site but diverged considerably further
upstream. Both promoters are active in mesenchymal but not epithelial
cells thus reflecting the expression pattern of the SF/HGF gene in
cells in vitro and in vivo. We have here identified
two regulatory sequences in the SF/HGF promoter: a negative element at
positions -239 to -258 and a positive element near the
major transcription start site; specific deletions destroyed the
activities of these elements. We were not able to localize elements on
the SF/HGF promoter region that mediate the previously described
effects of transforming growth factor ,
12-O-tetradecanoylphorbol-13-acetate, and coculture of
epithelial cells on SF/HGF gene expression. This study represents a
first step toward understanding the intricately regulated and cell
type-specific expression of the paracrine acting SF/HGF.
Mesenchymal-epithelial interactions are essential for organ
development and regenerative processes in vertebrates, and disturbances
of these interactions play a major role in various diseases including
cancer. The biology of mesenchymal-epithelial interactions have been
extensively studied, and it is recognized that a variety of mesenchymal
factors participate in the regulation of epithelial cell growth,
differentiation, and morphogenesis(1, 2) . Less is
known, however, about the molecular nature of the signals between
mesenchyme and epithelium; these may involve cell adhesion molecules,
components of the extracellular matrix, or secretory factors produced
by mesenchymal cells and acting on epithelia in a paracrine manner
(reviewed in (3) ). Among the latter, there exist several
ligands for epithelial receptor tyrosine kinases, e.g. scatter
factor/hepatocyte growth factor (SF/HGF), ()neuregulin (also
called neu differentiation factor, or heregulin) or keratinocyte growth
factor. All these factors are produced by mesenchymal cells, bind to
membrane receptors expressed in mainly epithelial cells (c-met, the
c-erbBs, and the keratinocyte growth factor receptor, 4-8), and
are potent effectors of epithelial growth, movement, and
differentiation in
vitro(5, 8, 9, 10) .
Scatter factor/hepatocyte growth factor, the specific ligand for the c-met receptor, is a 90-kDa secreted glycoprotein, which consists of disulfide-linked heavy (H) and light (L) chains generated by proteolytic cleavage from a single precursor molecule: the H-chain contains an N-terminal hairpin structure and four kringle domains, the L-chain is an inactive serine protease due to replacement mutations in 2 out of 3 resides of the catalytic site(11, 12, 13, 14, 15, 16, 17) . The structure of SF/HGF is thus similar to blood proteases such as plasminogen but not to other known ligands for receptor type tyrosine kinases. Two distinct activities, the ability to induce proliferation and movement of epithelial cells, have been used to independently isolate and molecularily characterize the factor(5, 10, 11, 12, 13, 14, 15, 16, 17) . SF/HGF can also increase invasiveness of epithelial cells and acts as a cytostatic factor on certain other cells(10, 18, 19) . In addition, SF/HGF is an inducer of epithelial tubulogenesis in vitro(20) : When Madin-Darby canine kidney epithelial cells are cultured in collagen gels together with SF/HGF, they rapidly proliferate and form complex networks of branching tubules. In situ hybridization analysis demonstrated that during mouse development, the c-met receptor is expressed in many epithelia whereas transcripts for the ligand SF/HGF are preferentially found in nearby mesenchymal cells(6) . SF/HGF also plays an important role in liver regeneration since in animals, plasma levels of the factor and cellular mRNA are elevated after partial hepatectomy or liver damage induced by hepatotoxins(21, 22, 23, 24) . Thus, SF/HGF and c-met constitute a paracrine signaling system, a concept originally proposed by Stoker and colleagues(5, 6, 25) , that is acting during organ development and regeneration. SF/HGF is also expressed in distinct embryonal muscle and brain cells(6) .
Little is
known about the regulation of SF/HGF expression in mesenchymal cells.
It has been shown that in MRC5 fibroblasts, SF/HGF expression is
down-regulated by TGF1 or by coculture with epithelial
cells(26, 27, 28) . In contrast, interleukin
1
, tumor necrosis factor-
, and the newly identified factor
injurin increased SF/HGF expression(29, 30) . In
primary human fibroblasts and HL60 leukemia cells, SF/HGF expression
was also stimulated by phorbol esters(31, 32) . The
sequences of the human and rat SF/HGF gene promoters have been
determined and the major transcription start sites were
mapped(33, 34) , but no activity studies have been
reported. In the present investigation, we show that sequences around
the major transcription start site of the mouse SF/HGF promoter are
sufficient to direct expression in fibroblasts but not in epithelial
cells and that a negative regulatory element is located between
positions -239 and -258 of the promoter.
Fragments
spanning the human promoter were cloned after PCR amplification using
5`-nested primers (35) and human placenta DNA (donated by Dr.
Ilse Wieland, University of Essen); 5` primer (h-991/-10179):
CTCCTGCAGGATTTCCGGTGAAAGTCAGTCCTAACC; 5` primer (h-345/-372):
CTCCTGCAGCTGCCTGTGCCTTGATTTAGCCATTGG; 3` primer (h+32/+58):
CCAGGCATCTCCTCCAGAGGGATCCGCTCTAGACTC. After digestion with the
restriction enzymes XbaI and PstI, the fragments were
cloned into Bluescript SK+ and pCAT-basic (Promega). The correct
sequence was confirmed by sequencing with T7 DNA polymerase (Pharmacia
Biotech Inc.). The human promoter CAT constructs had a common 3`-end at
position +58. Relative promoter activities of the various
constructs were estimated by comparison with the promoter activity of a
CAT plasmid containing the simian virus 40 promoter/enhancer CAT gene
(Promega). CAT assays were performed after transient transfection using
the calcium-phosphate method, and activities were quantified as
described(37) . A control plasmid containing the Rous sarcoma
virus promoter and the Escherichia coli lacZ gene was
cotransfected in each experiment. The amount of cell extracts used in
CAT assays was adjusted according to the -galactosidase
activities. Each transfection experiment was carried out twice with
double values and with two different preparations of the same plasmid.
To assess the effect of TGF
1 on promoter activity, transfected
cells were treated 14 h post-transfection with 5 ng/ml of the factor
(Boehringer Mannheim), and cell extracts were prepared 24 h later. To
analyze the effect of coculture with epithelial cells, equal numbers of
either mitomycin-treated Madin-Darby canine kidney cells or, as a
contol, mitomycin-treated fibroblasts were immediately seeded on top of
the transfectants. Cocultures were continued for 40 h after which cell
extracts were prepared. Mitomycin treatment was as described (27) .
Figure 1: Sequences of the mouse (m) and human (h) SF/HGF promoter. Genomic sequences are aligned in the region between nt -514 and +113 relative to the most proximal transcription start site of the mouse gene. The ATG codon marked by dots represents the translation start site; nucleotides marked by stars represent the identity between the two sequences. Arrows mark the major transcription start site of the human gene (33) and the identified transcription start site of the mouse gene, which corresponds to the major and minor start sites of the rat gene(34) .
Figure 2: Relative activities of human (h) and mouse (m) SF/HGF promoter CAT constructs. Specific transcript initiation in fibroblasts. CAT assays are shown for ras3T3 fibroblasts (A) and MCF7 breast epithelial (carcinoma) cells (B). The activities of various deletion constructs were compared with those of the SV40 promoter/enhancer construct and of pCAT basic. Numbers indicate the position of the 5`-end of the promoter fragment used, relative to the major transcription start site. The m-755 construct of the mouse promoter was also tested in the reverse orientation (m-755r). C, scheme of the experimental design for the RNase protection assay. The HindIII-PvuII fragment from the chimeric -150/+34 SF/HGF-CAT fusion gene was cloned into the HindIII-Sma sites of Bluescribe M13+ and linearized with BglII as indicated to generate the specific run-off transcript. Relative positions of transcriptional start sites are marked with upward-pointing arrows. Hybridization of the specific antisense probe to RNA of transient transfectants yielded protected fragments of 270, 220, and 200 nt. Fragments of 113 nt correspond to properly initiated transcripts of the cotransfected SV40 promoter/enhancer plasmid. D, RNase protection assay of various transfected constructs in ras3T3 or MCF7 cells as indicated above the slots. The size marker M is pBR digested with MSPII. The input lane (I) shows the T7 antisense probe. The protected fragments corresponding to transcripts initiated at either the SF/HGF or the SV40 promoter are indicated by arrows.
In order to confirm that the observed differences in CAT activity result from a transcriptional effect, we mapped by RNase protection chimeric CAT-mRNA transcripts in fibroblasts (ras3T3) and epithelial cells (MCF7) transiently transfected with the mouse SF/HGF promoter. The antisense riboprobe that we used recognizes the N-terminal part of the CAT gene and mouse SF/HGF promoter sequences up to position -70 (Fig. 2C). Cells were transfected with the SV40 promoter/enhancer-driven CAT gene alone or in combination with the SF/HGF promoter construct m-365. We detected three protected fragments in ras3T3 fibroblasts but none in epithelial cells (Fig. 2D). This indicates tissue-specific transcription from the SF/HGF promoter in agreement with the results obtained in the CAT assays. The major protected fragments of 200, 220, and 270 nucleotides in length correspond to transcript initiation sites that have been mapped for the rat SF/HGF gene(34, 42) .
Figure 3: Activity of progressive 5` deletions of the mouse SF/HGF promoter. On the left, the transfected chimeric deletion CAT constructs are displayed; the numbers indicate the length of the constructs with respect to the major transcription start site. On the right, relative CAT-activities of the constructs in fibroblasts (ras3T3) and epithelial cells (MCF7) in comparison to the SV40 promoter/enhancer are shown. Major and minor transcription start sites are indicated by the two arrows.
It has previously been shown that SF/HGF mRNA levels in
fibroblasts are modulated after treatment with TGF1, TPA, and
coculture with epithelial
cells(26, 27, 32, 38, 39, 40) .
We subjected ras3T3 fibroblasts, which were transiently transfected
with the various promoter deletion constructs, to treatment with
TGF
1, TPA, and coculture with Madin-Darby canine kidney epithelial
cells. These treatments did not lead to significant changes in the
amounts of promoter CAT activities and in the profile seen when
different deletion constructs were compared (data not shown).
Figure 4: Nuclear factor binding to the SF/HGF promoter by footprint analysis. Fragments of the mouse SF/HGF promoter were labeled at position -239 (left picture, non-coding strand) and -70 (right picture, coding strand) and subjected to DNaseI footprint analysis using nuclear extracts of ras3T3 fibroblasts and MCF7 epithelial cells (see ``Materials and Methods''). The lanes marked(-) indicate digestion in the absence of nuclear extract. The lanes G and G + A are Maxam-Gilbert sequence reaction products. The region specifically protected in fibroblasts (+14 to -7 and -21 to -70) and the region protected in both cell lines (-229 to -258) are schematically displayed on the left and right side, respectively.
The region around the major transcription start site was also examined by gel retardation analysis with nuclear extracts from various cell lines (Fig. 5). Using an oligonucleotide spanning positions -7 to +34 and nuclear extracts of fibroblasts (ras3T3 and NIH3T3), three major retarded complexes were detected (arrowheads). The formation of these complexes was competed by the unlabeled oligonucleotide but not by an oligonucleotide from positions +14 to +34 or by an unrelated oligonucleotide (E-Pal). Interestingly, extracts of MCF7 epithelial and neuro 2A cells did not form the complex of intermediate size (large arrowhead).
Figure 5: Nuclear factor binding to the region covering the transcription start site of the mouse SF/HGF promoter by gel retardation analysis: Difference between SF/HGF-producing and non-producing cell lines. A, schematic representation of the radiolabeled oligonucleotide probe (-7 to -34) used for gel retardation assays. B, gel retardation assay using nuclear extracts from ras3T3 fibroblasts and MCF7 epithelial cells. The specific competitor was the unlabeled -7 to +34 oligonucleotide, a second oligonucleotide was from position +14 to +34, and a nonspecific competitor was from the E-cadherin promoter (E-Pal, cf. (37) ). Unlabeled oligonucleotides were used at 50-fold molar excess. C, gel retardation assay using nuclear extracts from 3T3 fibroblasts and neuro 2A (neuroblastoma) cells. Conditions were as in B.
Figure 6: Deletion of the promoter regions presumed to be important for positive and negative regulation. A, the region -258 to -239 specifically protected in footprint analysis (cf.Fig. 4) was deleted from the m-291 construct, and B, the region -66 to +34 was deleted from the mouse -755 construct. C and D, CAT assays showing the effects of these two deletions. CAT activities of the constructs m-755, m-150, and pCAT basic are shown for comparison.
In the present investigation we examined, by promoter
analysis, the regulation of the scatter factor/hepatocyte growth factor
gene which is, in vivo, expressed in mesenchymal (and some
neuronal) but not in epithelial cells(6) . The target of SF/HGF
is the Met receptor tyrosine kinase which is predominantly produced by
epithelial and endothelial but not mesenchymal cells. We show here that
the activity of our SF/HGF promoter constructs is restricted to
mesenchymal cells, as shown by CAT and RNase protection assays, and we
have identified positive and negative regulatory elements in this
promoter fragment (Fig. 7). The positive regulatory elements are
located around the major transcription start site and upstream of
position -291, a negative regulatory element is located at
positions -239 to -258. Our work has failed to provide
evidence for a role of the IL6 and TGF response elements
previously identified in the human and rat promoter (cf. Refs.
33, 34).
Figure 7:
Functionally important sequences in the
SF/HGF promoter. Large (shadowed) boxes,
positive and negative regulatory regions identified by deletion and
footprint analysis. Arrows indicate major and minor
transcription start sites. HLH, NF1, hAPF1,
and AP-1 are consensus binding sites for helix-loop-helix
transcription factors, nuclear factor-1, human interleukin 6-dependent
transcription factor, and AP-1 transcription factor. TGF/TIE, TGF
inhibitory element (black
box); IL6RE, interleukin 6-responsive elements (
); NFIL6, interleukin 6-dependent nuclear factor (
). P1, P2, and P3 are palindromic sequences.
The negative regulatory element in the SF/HGF promoter was
uncovered due to a 3-4-fold drop of promoter activity in a 5`
deletion experiment and was also detected in our footprint analysis as
a region of factor binding (positions -258 to -229).
Indeed, internal deletion of the protected sequences fully released the
inhibitory effect of the element. This sequence, therefore, is a
negative regulatory element for which some preliminary evidence may
have been presented by others(41) . Interestingly, the sequence
contains putative binding sites for helix-loop-helix transcription
factors and nuclear factor-1 (42) as well as a previously
identified palindrome, P2(33) . However, this element appears
to be largely promoter context dependent since it had no negative
influence on the heterologous SV40 and TK promoters; to our surprise,
it stimulated the activity of the E-cadherin promoter when inserted at
position -78 (data not shown, cf.(37) ). A
promoter context-dependent element with similar characteristics has
also been described in the human erbB-2 promoter(43) .
Footprint analysis also uncovered nuclear factor binding sites to
regions close to the transcriptional start sites, which are specific
for extracts of mesenchymal cells. The 5` deletion analysis revealed
that the region between positions -7 and +14 is of
particular importance, and gel retardation experiments with an
oligonucleotide from this region showed the formation of a specific
proteinDNA complex which is characteristic for SF/HGF-expressing
cells. The sequence -7 to +14 of the SF/HGF promoter does
not fit the initiator sequence 5`-CTCANTCT-3` described for well
studied TATA-less promoters(44) . A noncanonical TATA element
(AATAAA) at position -24 might be responsible for transcription
initiation at multiple sites.
Much effort has here been undertaken
to identify elements in the SF/HGF promoter which reflect the effects
of various modulators of SF/HGF expression in vivo and in cell
culture, such as TGF(26, 38) , TPA(32) ,
or factors produced by cocultured epithelial cells(27) . None
of these factors significantly influenced promoter activity in our
transient transfection assays. This suggests that additional
cis-elements outside the promoter region we have studied here are
involved in the response to these agents or that the effect of
TGF
, TPA, and coculture with epithelial cells is translational
rather than transcriptional. Nuclear run-off transcription reactions
and measurements of the half-life of the SF/HGF transcript in
fibroblast cultures exposed to TGF
, TPA, or IL6 should clarify
this point. After completion of this work, a paper appeared (45) which describes aspects of the SF/HGF promoter.
Surprisingly, these authors found activity also in the carcinoma
(epithelial) cell line RL 95-2. No factor binding by footprinting
or bandshift analysis is shown. However, IL6 treatment stimulated
promoter activity 2.5-fold in a stably transfected cell clone. No
stimulation of SF/HGF expression by IL6 was reported by
others(29) .
Future experiments in transgenic animals will
show whether the SF/HGF promoter elements identified in the present
study are sufficient to generate the expression pattern as seen with
the endogenous gene. In particular, it will be interesting to examine
whether the SF/HGF cDNA driven by this promoter fragment can rescue the
lethal phenotype of mice with homozygous deletion of the SF/HGF gene. ()Furthermore, we will compare promoter fragments with and
without the negatively acting element in order to see in which tissues
this element may suppress SF/HGF expression in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X81630[GenBank].