(Received for publication, July 5, 1995; and in revised form, January 16, 1996)
From the
Transforming growth factor- (TGF-
) plays a major role
in regulating connective tissue deposition by controlling both
extracellular matrix production and degradation. In this study, we show
that TGF-
transcriptionally represses both basal and tumor
necrosis factor-
-induced collagenase (matrix metalloprotease-1)
gene expression in dermal fibroblasts in culture, whereas it activates
its expression in epidermal keratinocytes. We demonstrate that this
differential effect of TGF-
on collagenase gene expression is due
to a cell type-specific induction of distinct oncogenes of the Jun
family, which participate in the formation of AP-1 complexes with
different trans-activating properties. Specifically, our data
indicate that the inhibitory effect of TGF-
in fibroblasts is
likely to be mediated by jun-B, based on the following
observations: (a) TGF-
induces high levels of jun-B expression and (b) over-expression of jun-B mimics TGF-
effect in inhibiting basal collagenase promoter
activity and preventing tumor necrosis factor-
-induced trans-activation of the collagenase promoter. In contrast,
TGF-
induction of collagenase gene expression in keratinocytes is
preceded by transient elevation of c-jun proto-oncogene
expression. Over-expression of c-jun leads to trans-activation of the collagenase promoter in both cell
types, suggesting that c-jun is a ubiquitous inducer of
collagenase gene expression. Transfection of keratinocytes with an
antisense c-jun construct together with a collagenase
promoter/reporter gene construct inhibits basal and TGF-
-induced
up-regulation of the collagenase promoter activity, implying that c-jun mediates TGF-
effect in this cell type.
Collectively, our data suggest differential signaling pathways for
TGF-
in dermal fibroblasts and epidermal keratinocytes, leading to
cell type-specific induction of two AP-1 components with opposite
transcriptional activities.
Matrix metalloproteases comprise a family of proteolytic enzymes involved in the degradation of the extracellular matrix of connective tissue (for reviews see (1) and (2) ). These enzymes play a critical role in a number of physiological and pathological processes involving connective tissue remodeling and/or destruction, as exemplified by embryonic development, wound repair, tumor metastasis, and rheumatoid arthritis. Breakdown of the fibrillar collagen network is initiated by interstitial collagenase (matrix metalloprotease-1), whereas the other components of the matrix are degraded primarily by stromelysins and gelatinases.
The expression of matrix
metalloproteases by connective tissue cells is modulated by a variety
of cytokines and growth factors(2) . In particular,
interleukin-1 and tumor necrosis factor- (TNF-
) (
)are potent activators of fibroblast collagenase gene
expression, and their effect is mediated by c-Jun, the product of the c-jun proto-oncogene, which participates in the formation of
the transcription factor AP-1(1, 2) . In contrast,
transforming growth factor-
(TGF-
) has been shown to inhibit
fibroblast collagenase gene expression through Jun-B-dependent
mechanisms(3) , whereas it inhibits the expression of transin,
the rat homologue of stromelysin, through Fos-mediated
mechanisms(4) .
Recent in vivo observations have
revealed that during cutaneous wound healing, the expression of
collagenase is very low in the dermis, whereas it is markedly elevated
in basal keratinocytes at the wound edges(5) . In this context,
the close topographic proximity of fibroblasts and keratinocytes led us
to investigate in vitro the signals that would be responsible
for the differential, cell type-specific expression of collagenase
during wound healing. We report that TGF-, a growth factor with
essential wound healing promoting
activities(6, 7, 8, 9) , is a potent
inhibitor of collagenase gene expression in fibroblasts, whereas it
strongly up-regulates collagenase expression in keratinocytes. We
demonstrate that cell-specific induction of different oncogenes of the
Jun family, with opposite trans-activating properties, is
responsible for the differential regulation of collagenase gene
expression by TGF-
in fibroblasts and keratinocytes.
Human epidermal keratinocytes obtained by explanting foreskin specimens, were grown in serum-free, low calcium (0.15 mM), keratinocyte growth medium supplemented with epidermal growth factor, hydrocortisone, insulin, and bovine pituitary extract (KGM, Clonetics Corp., San Diego, CA), and utilized in passage 1 to avoid differentiation inherent to subculturing of these cells. One hour prior to the experiments, the confluent keratinocyte cultures were placed in fresh KGM.
To study the
transcriptional regulation of collagenase gene expression, the
following plasmid construct was used in transient transfection
experiments: pCLCAT3, which contains 3.8 kilobase pairs of
5`-flanking DNA of human collagenase gene linked to the CAT reporter
gene(15) , kindly provided by Dr. Steven M. Frisch (La Jolla
Cancer Research Foundation, La Jolla, CA). The oncogene expression
vectors described above were used in co-transfection experiments. Empty
pRSVe was used as filling plasmid in order to transfect the same amount
of DNA in every cell plate.
To prepare an antisense c-jun construct, a fragment spanning the region +451 to +617 of the c-jun gene was amplified by polymerase chain reaction (PCR). The PCR amplimers were cloned into a PCRII plasmid vector (InVitrogen Corp., San Diego, CA), and the clones containing the insert were sequenced to ensure the fidelity of the PCR amplification. The PCR products were then inserted in an antisense orientation as XhoI-HindIII fragments into the pRSVe expression vector in order to generate the construct pRSV-ASc-jun.
Figure 1:
Effect of TGF- on collagenase gene
expression in dermal fibroblasts. Confluent fibroblast cultures were
incubated in medium containing 1% fetal calf serum without(-) or
with (+) TNF-
(10 ng/ml), in the absence(-) or the
presence (+) of TGF-
(10 ng/ml). After 24 h, total RNA was
extracted and analyzed by Northern hybridization with a
collagenase-specific cDNA; a GAPDH cDNA was used as a control. A, autoradiograms. B, densitometric analysis after
correction for GAPDH mRNA levels.
In
the second set of experiments, confluent keratinocyte cultures were
incubated for 24 h without or with TGF- and/or TNF-
at
concentrations of either 1 or 10 ng/ml. Contrasting with its inhibitory
effect observed in dermal fibroblasts, TGF-
was found to be a
potent enhancer of keratinocyte collagenase gene expression (Fig. 2, second and third lanes). On the other
hand, TNF-
, which is a potent enhancer of collagenase gene
expression in fibroblasts (see Fig. 1), had little effect on the
basal expression of collagenase in keratinocytes (Fig. 2, fifth and sixth lanes) and a minimal effect on the
induction exerted by TGF-
(Fig. 2, fourth lane).
Quantitation of the autoradiograms after normalization of collagenase
mRNA levels against those of GAPDH showed a 4.5-5-fold elevation
of collagenase mRNA steady-state levels upon TGF-
stimulation,
whereas TNF-
did not stimulate collagenase expression (Fig. 2B).
Figure 2:
Effect of TGF- on collagenase gene
expression in epidermal keratinocytes. Confluent keratinocyte cultures
in serum-free KGM were incubated for 24 h in the absence or the
presence of TGF-
without or with TNF-
in the concentrations
indicated. After 24 h, total RNA was extracted and analyzed by Northern
hybridization with a collagenase-specific cDNA; a GAPDH cDNA was used
as a control. A, autoradiograms. B, densitometric
analysis after correction for GAPDH mRNA
levels.
Additional experiments with various
concentrations (0.1, 1, and 10 ng/ml) of TGF- indicated a
stimulatory effect with as little as 0.1 ng/ml (
2.8-fold); whereas
maximal elevation of collagenase mRNA levels (
6-fold) was observed
with 1 ng/ml of TGF-
, no further enhancement was noted with 10
ng/ml of TGF-
(not shown).
Transient cell transfections with a
collagenase promoter/CAT reporter gene construct were performed to
examine whether TGF- regulates collagenase mRNA steady-state
levels through modulation of transcription at the promoter level. Human
neonatal fibroblasts were transfected with the collagenase promoter/CAT
construct pCLCAT3 and treated with TGF-
or TNF-
both at 10
ng/ml concentration. Assay of CAT activity after 40 h of incubation
indicated that TGF-
reduced the promoter activity by 40-60%,
as compared with that of control cultures (Fig. 3). Also
TGF-
counteracted TNF-
-induced elevation of the collagenase
promoter activity.
Figure 3:
Effect of TGF- on collagenase
promoter activity in dermal fibroblasts. Fibroblasts in late
logarithmic growth phase were transfected with 10 µg/plate of
collagenase promoter/CAT (pCLCAT3) plasmid construct as described under
``Materials and Methods.'' Following the glycerol shock, the
cells were placed in medium supplemented with 1% fetal calf serum.
Three hours later, TNF-
and/or TGF-
, both at a concentration
of 10 ng/ml, were added (+) and incubations were continued for 40
h. CAT activity, representing collagenase promoter activity, was
determined. The results, presented as relative promoter activity, are
the means ± S.D. of four independent experiments, each point run
with duplicate samples and expressed as fold induction over the
controls, which are set as 1.0.
In another set of experiments, confluent
keratinocyte cultures were transfected with the same collagenase
promoter/CAT reporter gene construct and treated with various doses of
TGF- (0.1, 1, and 10 ng/ml) for 40 h. Assay of CAT activity
revealed a dose-dependent elevation of the promoter activity (Fig. 4) with a maximal stimulation (
4-fold) observed with
10 ng/ml of TGF-
.
Figure 4:
Effect of TGF- on collagenase
promoter activity in epidermal keratinocytes. Confluent keratinocyte
cultures in serum-free KGM were transfected with pCLCAT3 using a
liposome-based method (DOTAP, Boehinger Mannheim) according to the
manufacturer's protocol. Five hours after transfections, the
medium was replaced with fresh KGM and TGF-
in various
concentrations was added 2 h later for a 40-h incubation. CAT activity,
representing collagenase promoter activity, was determined. The
results, presented as relative promoter activity, are the means
± S.D. of three independent experiments run with duplicate
samples and expressed as fold induction over the controls, which are
set as 1.0.
Thus, the enhancement of collagenase gene
expression in keratinocytes by TGF- and its inhibition in
fibroblasts, as detected at the mRNA level, was mediated, at least in
part, by cell was type-specific modulation of the promoter activity.
As shown in Fig. 5, TNF- rapidly (within 1 h of incubation) enhanced
the expression of c-jun in dermal fibroblasts (lane
4), and the induction persisted even after 6 h of incubation (lane 5). Also, the steady-state levels of jun-B mRNAs were elevated but to a lesser extent than those for c-jun (lane 4). TGF-
alone did not affect either
the basal expression of c-jun (lane 7) or the
induction of c-jun by TNF-
(lanes 9 and 10
versus lanes 4 and 5). In contrast, TGF-
, either
alone or in combination with TNF-
, strongly elevated the
expression of jun-B (lanes 7 and 9), which
persisted for at least up to 6 h following the initiation of
stimulation (lanes 8 and 10). It appears, therefore,
that in fibroblasts, TGF-
counteracts TNF-
-induced
collagenase gene expression, but this effect is not due to repression
of c-jun transcription. At the same time, TNF-
did not
alter the expression of jun-B induced by TGF-
.
Accordingly, TGF-
stimulation led to a dramatic reduction of the c-jun/jun-B mRNA ratio in fibroblasts, from 1.7 to
0.1 and 0.2 after 1 and 6 h of stimulation, respectively (Fig. 6). In contrast, TNF-
elevated the c-jun/jun-B mRNA ratio due to a substantially higher
stimulation of c-jun versus jun-B. When TGF-
was added
simultaneously with TNF-
, the c-jun/jun-B mRNA
ratio remained at levels that were
60-70% lower than
observed with TNF-
alone (Fig. 6). Therefore, there is a
strong correlation between the c-jun/jun-B mRNA ratio
and the level of collagenase expression, suggesting that the
modification of the ratio of c-jun/jun-B mRNA is
likely to reflect a reduction of the relative amounts of c-Jun versus those of Jun-B when TGF-
is present, leading to
reduced collagenase gene transcription.
Figure 5:
Effect of TNF- and TGF-
on c-jun and jun-B gene expression in dermal
fibroblasts. Confluent fibroblast cultures were incubated in medium
containing 1% fetal calf serum without (CTL) or with TNF-
(10 ng/ml), in the absence(-) or the presence (+) of
TGF-
(10 ng/ml). Total RNA was extracted after 0, 1, or 6 h of
incubation and analyzed by Northern hybridizations with
P-labeled cDNA probes specific for c-jun and jun-B mRNAs. A GAPDH cDNA was used as a
control.
Figure 6:
Effect of TGF- on the ratio of c-jun/jun-B mRNA in dermal fibroblasts. Relative c-jun and jun-B mRNA levels were determined in each
RNA preparation by densitometric analysis of the autoradiograms shown
in Fig. 5. Raw values were corrected for GAPDH mRNA levels in
the same RNA preparations and for the specific activity of the c-jun and jun-B probes. The ratio of c-jun and jun-B mRNA levels was calculated for the various time
points of cytokine treatment. Open squares, control; solid
squares, TGF-
; open triangles, TNF-
; solid
triangles, TNF-
+ TGF-
.
Subsequently, we tested the
pattern of expression of oncogenes of the Jun family in the presence of
TGF- in epidermal keratinocytes. Contrasting with its lack of
effect in fibroblasts, TGF-
induced high levels of c-jun expression in keratinocytes, with a maximum enhancement at 1 h
following growth factor stimulation (Fig. 7). The high levels of c-jun mRNAs persisted at least 6 h post-stimulation and
preceded the induction of collagenase expression. In contrast, the
basal expression of jun-B was very low and, although
expression was enhanced after 1 h of incubation with TGF-
, it
remained well below the level of expression of c-jun. In fact,
using
P-labeled cDNA probes with similar specific
activities (
2
10
cpm/µg), a 48-h exposure
of the autoradiogram was necessary to detect the low jun-B expression, whereas a 14-h exposure was sufficient to easily
detect c-jun expression (Fig. 7). Therefore, in
keratinocytes, after TGF-
stimulation, the ratio c-jun/jun-B is largely in favor of c-jun,
25-fold more c-jun mRNA than jun-B at 1 h after
addition of TGF-
, as estimated by scanning densitometry of the
autoradiograms, correction for GAPDH mRNA levels in the RNA
preparations, and differences in the exposure times of the
autoradiograms. These data contrast the reverse situation observed in
dermal fibroblasts in which jun-B expression is boosted by
TGF-
treatment (see above, Fig. 5and Fig. 6). It
should be noted that the extent of stimulation of collagenase gene
expression as detected at the mRNA level (
15-fold after 6 h) was
more pronounced than the induction of promoter activity observed in
transient cell transfection experiments (see Fig. 4). It is
conceivable that TGF-
, in addition to activating the collagenase
promoter, may also increase collagenase gene expression by
post-transcriptional mechanisms such as stabilization of the
corresponding mRNA, as described previously for phorbol esters or
epidermal growth factor(20, 21) .
Figure 7:
Effect of TGF- on c-jun and jun-B gene expression in epidermal keratinocytes.
Confluent keratinocyte cultures were incubated in serum-free KGM with
TGF-
(10 ng/ml). Total RNA was extracted at various time points
following the addition of TGF-
and analyzed by Northern
hybridizations with cDNAs specific for collagenase and c-jun and jun-B mRNAs. A GAPDH cDNA was used as a control. The
specific activities of the probes,
2
10
cpm/µg, differed by less than 15%. Exposure times of the
different hybridizations were 14 h for c-jun, 48 h for jun-B, and 24 h for collagenase.
Figure 8:
Characterization of pRSV-ASc-jun in stable transfection experiments. In order to verify the
activity of the pRSV-ASc-jun antisense construct, pRSVeNIH3T3
and pRSV-ASc-junNIH3T3 fibroblast cultures were grown to
confluency. Three hours prior to the addition of growth factors, the
confluent fibroblast cultures were rinsed with DMEM and placed in DMEM
containing 1% fetal calf serum. The cultures were then treated for 6 h
with either TNF- or TGF-
, each at a concentration of 10
ng/ml. Total RNA was extracted and analyzed by Northern hybridization
with
P-labeled cDNA probes for collagenase, c-jun and jun-B. A GAPDH cDNA was used as a
control.
Figure 9:
Effect of antisense c-jun on
TGF--mediated up-regulation of collagenase promoter activity in
keratinocytes. Confluent keratinocyte cultures were transfected with 3
µg/plate of pCLCAT3, together with 17 µg/plate of either pRSVe
or pRSV-ASc-jun. After 18 h, medium was replaced with fresh
KGM. Six hours later, TGF-
(10 ng/ml) was added to the cultures
(+). Incubations were continued for 40 h, and CAT activity,
representing the promoter activity, was determined. The results are the
means ± S.D. of three independent
experiments.
Several studies have shown differences in the pattern of expression and response to extracellular stimuli between c-jun and jun-B in a variety of experimental systems(3, 22, 23, 24) . Furthermore, considerable differences in their trans-activation and transforming activities have been reported(3, 14, 25, 26) . For example, whereas c-Jun is an efficient activator of the c-jun and collagenase promoters that contain a single TRE, Jun-B is not. In addition, Jun-B counteracts activation of these promoters by c-Jun. However, like c-Jun, Jun-B is a potent activator of constructs containing multimeric TREs. These differences in the biological actions of the two Jun proteins are due to intrinsic differences in their activation and DNA-binding domains (14, 27) , allowing fine tuning of the regulation of TRE-driven genes.
The regulatory role of the different Jun proteins is further emphasized by the fact that the corresponding genes are not coordinately expressed in different tissues, as shown in adult mice and during embryogenesis (24, 28, 29) , suggesting a tissue-specific transcriptional regulation of TRE-driven target genes. In this context, it has been recently shown that jun-B and c-jun are selectively up-regulated and functionally implicated in the development of fibrosarcoma(30) . It is therefore conceivable that in different inducible systems, increased specificity and precise regulation of TRE-driven transcriptional activation is achieved by interactions between positive and negative transcription factors that belong to the same gene family.
In this study, we have provided the
following evidence for a mediation of the inhibitory effect of
TGF- on fibroblast collagenase gene expression by Jun-B: (a) TGF-
induces high levels of jun-B expression; (b) jun-B expression vectors mimic
TGF-
action in our experimental system by inhibiting basal
collagenase promoter activity and by exerting an antagonistic effect on
TNF-
-induced collagenase gene expression. On the other hand, we
have demonstrated that TGF-
stimulates collagenase gene expression
in keratinocytes and that this effect is mediated by c-jun, as
follows: (a) TGF-
induces high levels of c-jun expression; (b) c-jun expression vectors trans-activate the collagenase promoter; (c)
antisense c-jun expression vectors prevent TGF-
activation of collagenase gene expression. A schematic diagram
depicting the differential effects of TGF-
on collagenase gene
expression in fibroblasts and keratinocytes is shown in Fig. 10.
These results are the first evidence of differential induction of two
oncogenes with opposite trans-activation properties upon
stimulation by a single growth factor, TGF-
, in two different cell
types within the same tissue, the skin.
Figure 10:
Schematic representation of the putative
mechanisms for differential regulation of collagenase gene expression
by TGF- in fibroblasts and keratinocytes. Left,
TGF-
, through interactions with specific receptors on the
keratinocyte surface, induces high levels of expression of c-jun, which, after dimerization, is responsible for the trans-activation of the collagenase promoter. Right,
TGF-
induces high levels of expression of jun-B, and its
product participates in the formation of AP-1 complexes with inhibitory
activity on collagenase gene expression. Transduction mechanisms (1; to be identified) induce transcription of c-jun in keratinocytes and of jun-B in fibroblasts (2). Jun products are translated in the cytoplasm (3)
and translocate into the nucleus (4) to form AP-1 complexes
that modulate collagenase gene transcription (5).
TGF-
-induced c-jun expression in keratinocytes leads to
increased collagenase gene transcription (+) and increased
collagenase production (&cjs3832;&cjs3832;). In fibroblasts, increased jun-B expression represses collagenase transcription(-)
with subsequent decrease in collagenase production
(&cjs3706;&cjs3706;).
TGF- has been shown to
reduce collagenase gene expression and activity in cultured
fibroblasts. This inhibition results from two distinct mechanisms: (a) TGF-
reduces the expression of the collagenase gene
and (b) the expression of tissue inhibitor of metalloproteases
is elevated by TGF-
(31) . Our data indicate that the
concept of TGF-
as a potent inhibitor of matrix remodeling is cell
type-specific because this growth factor is a potent activator of
collagenase gene expression in epidermal keratinocytes in culture. In
that respect, TGF-
has been shown previously to up-regulate both
92- and 72-kDa gelatinase activity and gene expression in both
fibroblasts and keratinocytes(32, 33) .
Recently,
it has been demonstrated using in situ hybridization in
ulcerative skin lesions such as pyogenic granulomas that collagenase is
expressed near the advancing edge of the ulceration, within the
disrupted epidermis adjacent to an ulcer(5) . By contrast, no
hybridization signal was detected within the dermis or normal, intact
epidermis. Therefore, basal keratinocytes seem to be the primary source
of collagenase during wound healing, suggesting that keratinocytes play
an essential role in tissue remodeling. It has been suggested that the
signals that activate collagenase in keratinocytes are provided by the
dermal extracellular matrix. In agreement with this hypothesis is the
fact that keratinocytes grown on type I collagen exhibit enhanced
collagenase production(34) . By contrast, activation of
fibroblast collagenase expression may be mediated by soluble factors
such as interleukin-1 or TNF-, rather than by the extracellular
matrix. Skin injury is accompanied by release of interleukin-1, which
in turn may activate fibroblasts but not keratinocytes to produce
collagenase(34) . Our data provide an alternative model for the
cell-specific activation of collagenase gene expression during wound
healing in which TGF-
, which is present in abundant amounts in the
healing wound bed, could simultaneously turn off the expression of
collagenase in fibroblasts while activating that of keratinocytes
directly in contact with the dermis. We hypothesize that
collagenase-secreting keratinocytes, possibly in response to TGF-
,
may be able to migrate to close the wound. This hypothesis is supported
indirectly by a previous study indicating that TGF-
stimulates the
outgrowth of epidermal cells from skin explant cultures(35) .
In conclusion, this study has provided the first evidence for cell
type-specific, differential induction of two transcription factors of
the same family with antagonistic trans-activation properties,
leading to opposite regulation of collagenase gene expression in
fibroblasts and keratinocytes by TGF-.