(Received for publication, October 30, 1995; and in revised form, February 9, 1996)
From the
A series of studies has shown that application of transforming
growth factor (TGF-
) to a wound has a beneficial effect,
especially in animals with wound healing disorders. In this study we
have investigated the regulation of TGF-
1,
2, and
3 and
their receptors during the repair process. We found a large induction
of all three TGF-
isoforms and also of TGF-
types I and II
receptors, although the time course of induction and the absolute
expression levels were different for these genes. Furthermore, each
TGF-
isoform had distinct sites of expression in the wound.
Systemic treatment with glucocorticoids significantly altered the
expression levels of TGF-
s and TGF-
receptors. Whereas
expression of TGF-
1, TGF-
2, and TGF-
type II receptor
was suppressed by glucocorticoids in normal and wounded skin,
expression of TGF-
3 and TGF-
receptor type I mRNA was
stimulated. These findings provide an explanation for the beneficial
effect of exogenous TGF-
in the treatment of impaired wound
healing in glucocorticoid-treated animals. Furthermore, they suggest
that a disturbed balance between the levels of the three TGF-
isoforms and their receptors might underlie the wound healing defect
seen in glucocorticoid-treated animals.
Wound healing is a highly ordered and well coordinated process
that involves inflammation, cell proliferation, matrix deposition, and
tissue remodeling(1) . A series of studies indicates that
peptide growth factors and their receptors are key modulators of this
process. One of the most important modulators of wound repair is
transforming growth factor (TGF-
), (
)which is
found in large amounts in platelets(2) . Furthermore, it is
produced by several cell types that are present in a wound, including
activated macrophages, neutrophils, fibroblasts, and also
keratinocytes(3, 4, 5, 6, 7, 8, 9) .
Three TGF-
isoforms (TGF-
1,
2, and
3) are present
in mammals that share a 64-85% amino acid sequence
homology(10) . In vitro, TGF-
s have potent
effects on many cell types. Thus, they are mitogenic for various types
of fibroblasts, but they inhibit proliferation of most other cells,
including endothelial cells and epithelial cells. Furthermore,
TGF-
s modulate differentiation processes, and they are very potent
stimulators of the expression of extracellular matrix proteins and
integrins(7, 10) . Therefore, they have the properties
expected of wound cytokines. Indeed, all three types of mammalian
TGF-
are expressed during wound repair, whereby each member of the
TGF-
family has a unique distribution in pig wounds(8) .
The differential expression of TGF-
s in the wound and also in many
embryonic mouse tissues (11, 12) suggested
differential regulation of these genes but also different functions of
their gene products. This was confirmed in in vitro studies
where several differences in the potency and biological activity of
TGF-
1,
2, and
3 have been demonstrated. These include
the inhibitory effect on DNA synthesis in keratinocytes, where
TGF-
3 is significantly more potent than TGF-
1 and
TGF-
2(13) . Recently Shah et al.(14) were able to demonstrate differences in the
biological effects of TGF-
s during wound repair. Their studies
suggest that TGF-
1 and TGF-
2 induce cutaneous scarring,
whereas TGF-
3 seems to inhibit this effect.
Whereas increased
expression of TGF-1 and TGF-
2 might have deleterious effects
on the repair process by increasing scar formation, reduced expression
of growth factors in a wound might result in a severe delay in wound
healing. This has recently been demonstrated for keratinocyte growth
factor, a member of the fibroblast growth factor family, which is
expressed at significantly reduced levels during wound repair in
genetically diabetic db/db mice (15) and
glucocorticoid-treated mice(16) . The wound healing defect seen
in these animals can be reversed by topical application of exogenous
growth factors. Thus, treatment of poorly healing wounds in
glucocorticoid-treated animals with fibroblast growth factors or
TGF-
had favorable effects on the repair
process(17, 18, 19, 20) . Therefore,
we speculated that expression of endogenous TGF-
s might also be
impaired in these animals. Here we demonstrate severe effects of
glucocorticoids on the expression of TGF-
s in normal and wounded
skin. These data suggest that aberrant expression of these genes in
glucocorticoid-treated mice might be associated with the wound healing
defect seen in these animals.
Figure 1:
Expression of TGF-s and TGF-
receptors in the dermis and the epidermis of mouse tails. Mouse tail
skin was incubated for 30 min at 37 °C in a solution of 2 M NaBr. The epidermis was subsequently separated from the underlying
dermis, immediately frozen in liquid nitrogen, and used for RNA
isolation. Fifty µg of RNA from dermis and epidermis was
subsequently analyzed by RNase protection assay for the expression of
TGF-
s and TGF-
receptors. One thousand cpm of the
hybridization probes was used as a size marker. The same batch of RNAs
was used for all protection assays. RI and RII, types
I and II receptor, respectively.
Figure 2:
Differential expression of TGF-1,
TGF-
2, and TGF-
3 mRNA during wound repair. Total cellular RNA
(50 µg) from normal and wounded back skin was analyzed by RNase
protection assay with RNA hybridization probes complementary to mRNA
encoding TGF-
1 (panel A), TGF-
2 (panel B),
and TGF-
3 (panel C). The same RNA preparations were used
for all hybridizations of this figure and Fig. 5. The time after
injury is indicated at the top of each lane. One
thousand cpm of the hybridization probes was used as the size marker.
The degree of TGF-
1,
2, and
3 mRNA induction as assessed
by laser scanning densitometry of the autoradiograms is shown
schematically in panel D.
Figure 5:
Glucocorticoids have differential effects
on TGF-1,
2, and
3 expression in normal skin. BALB/c
mice were treated with glucocorticoids as described under
``Materials and Methods.'' Nonwounded back skin from
dexamethasone-treated mice (skin + Dex) and control mice (control skin) was isolated and used for RNA isolation. Fifty
µg of total cellular RNA was analyzed by RNase protection assay for
expression of TGF-
1,
2, and
3 mRNA. One thousand cpm of
the hybridization probes was used as a size marker. The same set of
RNAs was used for the three protection assays shown in this
figure.
To determine the absolute levels of the
individual TGF- transcripts, defined amounts of the corresponding
sense transcripts were used as positive controls and compared with the
signals obtained with 20 µg of wound RNA. All sense transcripts had
the same length and were derived from homologous regions of the
TGF-
cDNAs. These results revealed a similar expression of
TGF-
1 and
3 at later stages of the repair process (5-7
days after injury), whereas TGF-
2 mRNA levels were 90% lower (data
not shown). Since TGF-
3 expression is significantly lower during
the early phase of wound healing (days 1-3 after injury) (Fig. 2), TGF-
1 seems to be the predominant isoform during
this period.
To determine the
localization of the different types of TGF- in normal and wounded
skin, serial sections from 5-day full-thickness mouse wounds were
stained with monospecific antibodies against TGF-
1,
2, and
3. At that time point after injury, all types of TGF-
are
expressed at high levels ( Fig. 2and Fig. 3). As shown in Fig. 3, A-C, TGF-
1,
2, and
3
proteins were found at distinct places within the wound. A remarkably
high expression of all three isoforms was seen in a population of cells
in the dermis at the wound edge, which might either represent migrating
fibroblasts or macrophages. Furthermore, all three isoforms were found
in the granulation tissue, whereas TGF-
2 protein was particularly
abundant below the hyperproliferative epithelium (Fig. 3B). TGF-
3 protein was found at high levels
in the hyperproliferative epithelium at the wound edge (Fig. 3C) but not in normal epidermis (data not shown).
By contrast, only a weak expression of TGF-
1 protein was seen in
differentiating keratinocytes of the hyperthickened epidermis (Fig. 3A), and TGF-
2 protein was restricted to
differentiated keratinocytes of the uppermost layers of the epithelium (Fig. 3B). These results demonstrate a differential
expression of all three TGF-
isoforms in the wound tissue.
Figure 3:
Differential expression of TGF-1,
2, and
3 proteins in 5-day mouse wounds. Frozen serial
sections from a 5-day mouse wound were incubated with monospecific
antibodies directed against TGF-
1 (panel A), TGF-
2 (panel B), or TGF-
3 (panel C) and stained with
the avidin-biotin-peroxidase complex system using
3-amino-9-ethylcarbazole as a chromogenic substrate. Nuclei were
counterstained with hematoxylin. An overview of the complete wound is
shown in panels A-C. G, granulation tissue; HE, hyperproliferative epithelium; M, muscle
layer.
Figure 4:
TGF- type I and type II receptors are
expressed at high levels in normal and wounded skin. Panel A,
50 µg of total cellular RNA from normal and wounded back skin was
analyzed by RNase protection assay for expression of TGF-
type I (TGF-
RI) and type II (TGF-
RII) receptors.
The time after injury is indicated at the top of each lane. One thousand cpm of the hybridization probes was used as
a size marker. One µg of the same batch of RNA is shown in panel B. The same RNA preparations were used for the
protection assays shown in Fig. 2and Fig. 5.
In nonwounded
skin, expression levels of TGF-1 and TGF-
2 were reduced by
dexamethasone (Fig. 5, A and B), whereas
TGF-
3 mRNA levels increased upon glucocorticoid treatment (Fig. 5C). These differences were also seen after
injury, and expression levels of TGF-
1 and TGF-
2 were 65%
lower in glucocorticoid-treated mice compared with control mice at day
3 after wounding (Fig. 6, A and B). A similar
reduction was seen at day 2 and 5 after injury (Fig. 6, A and B, and data not shown). In contrast to TGF-
1 and
TGF-
2, expression of TGF-
3 mRNA was stimulated by
dexamethasone, although this difference was only seen in the early
phase of wound repair when TGF-
3 mRNA expression is normally very
low (Fig. 6C). Thus, systemic treatment with
glucocorticoids resulted in a premature onset of TGF-
3 expression.
These in vivo data demonstrate that glucocorticoids modulate
the normal induction of TGF-
1,
2, and
3 expression after
cutaneous injury.
Figure 6:
TGF-1, TGF-
2, and TGF-
3 are
differentially regulated by glucocorticoids during wound healing.
BALB/c mice were treated with glucocorticoids as described under
``Materials and Methods.'' Mice injected with
phosphate-buffered saline were used as a control. RNA was isolated from
nonwounded skin of control mice (control skin) and from
wounded skin (3 and 5 days after wounding) of control mice and
dexamethasone-treated mice (+Dex). Fifty µg of total
cellular RNA from two independent experiments (experiments 1 and 2) was
analyzed by RNase protection assay for TGF-
1 (panel A),
TGF-
2 (panel B), and TGF-
3 (panel C)
expression. One thousand cpm of the hybridization probes was used as a
size marker. The degree of TGF-
1,
2, or
3 mRNA induction
as assessed by laser scanning densitometry of the autoradiograms is
shown schematically on the right side of the figure. The same
set of RNAs was used for the three protection assays shown in this
figure.
Figure 7:
Modulation of TGF- receptor
expression by glucocorticoids in nonwounded and wounded skin. BALB/c
mice were treated with glucocorticoids as described under
``Materials and Methods.'' RNA was isolated from nonwounded
back skin of control mice (control skin), from 2-day wounds of
control mice (2d wound control) and from mice treated with two
different concentrations of dexamethasone (0.2 or 1 mg/kg body weight) (2d wound + low Dex, 2d wound + high Dex). Fifty
µg of total cellular RNA was analyzed by RNase protection assay for
expression of TGF-
type I and II receptors (TGF-
RI and TGF-
RII, respectively). One thousand cpm of the
hybridization probes was used as a size
marker.
Wound healing is a highly organized process that is regulated
by a wide variety of growth factors and cytokines. Thus, the regulation
of the temporal and spatial expression of these factors is of major
significance for normal repair. One of the key players in the repair
process is TGF-. Expression of different TGF-
isoforms in
rabbit, porcine, and human wounds has been demonstrated by
immunohistochemical staining, yet with variable
results(8, 9, 28, 29, 30) .
Although these studies provide important information on the spatial
distribution of these factors in the wound, little is known about the
time course of TGF-
expression during the repair process. In this
study we demonstrate a strong up-regulation of TGF-
1,
2, and
3 expression after injury. These findings correlate with results
from other authors who demonstrated increased TGF-
-like activity
in wound fluid from rats(31) .
The kinetics of expression
was different for the three TGF- variants. Particularly remarkable
was the strong induction of TGF-
3 expression at later stages of
the repair process. Since TGF-
3 has been shown to reduce
connective tissue deposition and subsequent scarring during wound
healing in normal rats(14) , this finding suggests that
up-regulation of this factor after completion of the proliferative
phase of wound healing might be important for the limitation of the
fibrotic process. However, studies from other authors have yielded
contradictory results concerning the effect of TGF-
3 on connective
tissue deposition. They demonstrated an increase in new dermal matrix
by exogenous application of TGF-
3 to wounds in age-impaired animal
models(32) . However, the effect of TGF-
3 on scarring was
not determined in this study, and increased production of dermal matrix
during early wound repair might not necessarily lead to increased scar
formation. Besides the effects on the mesenchyme, TGF-
s are likely
to be important in the epidermis. TGF-
3 is the most abundant
TGF-
isoform in the hyperproliferative epithelium and might
therefore play an important role in keratinocyte differentiation. Thus,
increased expression of TGF-
3 during the early phase of wound
healing in glucocorticoid-treated mice could lead to a premature onset
of keratinocyte differentiation and inhibition of epithelial cell
proliferation. This could provide an explanation for the severe delay
in reepithelialization seen in these animals.
The distribution of
TGF-1,
2, and
3 in normal and wounded mouse skin showed
several differences compared with the expression pattern of these
factors in porcine and human skin, particularly in the epidermal
compartment(8, 29) . Thus, TGF-
2 was found at
high levels in all layers of the hyperproliferative epithelium of pig
wounds(8) , whereas in mouse wounds only the outermost layers
expressed this protein (this study). In normal murine epidermis,
TGF-
3 mRNA and protein were not detectable, whereas expression of
this factor was found at high levels in porcine and human
skin(8, 29) . This difference may be related to the
different rate of epidermal cell turnover in murine and human skin. In
contrast to TGF-
2 and TGF-
3, the expression pattern of
TGF-
1 in the epidermis was similar in all species, and TGF-
1
protein was found at highest levels in the upper layers of the
epidermis.
Recently, two different TGF- receptors have been
cloned which mediate TGF-
signal transduction. However, expression
of these receptors in a wound has not yet been demonstrated. Here we
show that both types of TGF-
receptor are expressed at high levels
in normal and wounded skin, suggesting that these receptors are indeed
mediators of TGF-
action in the wound. Interestingly,
glucocorticoids had differential effects on the expression levels of
both receptors, and the ratio of TGF-
receptor type I to type II
was increased significantly by steroid treatment. Recent studies had
suggested that the ratio of type I to type II receptor could influence
the biological effect of TGF-
s on proliferation and target gene
expression(33, 34) . Thus, increased expression of the
type I receptor as seen during wound healing in glucocorticoid-treated
mice might alter the cellular responses to the ligands during the
repair process.
The strong up-regulation of TGF-s during wound
healing suggested that defects in the regulation of their expression
might be associated with wound healing defects. This is supported by
the beneficial effect of exogenous TGF-
in the treatment of
impaired wound healing as seen, for example, in glucocorticoid-treated
mice(17, 19, 20) . Glucocorticoids are potent
anti-inflammatory agents that both stimulate and inhibit the
transcription of a variety of genes(35) . The influence of
glucocorticoids on wound healing is particularly remarkable, and the
prolonged administration of anti-inflammatory steroids leads to a delay
in wound repair and an increase in local wound
complications(36) . This wound healing defect may be due to the
suppression of the inflammatory phase of healing by inhibiting
leukocyte and macrophage infiltration (37, 38, 39) but also to the direct
inhibition of genes that play a role in the repair process. Thus, a
negative regulation by glucocorticoids has been shown for collagen type
I and tenascin mRNA expression(40, 41) . Furthermore,
keratinocyte growth factor expression in fibroblasts is suppressed by
dexamethasone(16) .
The beneficial effect of exogenous
TGF- on wound healing in glucocorticoid-treated animals (17, 19, 20) suggested that endogenous
TGF-
might also be limited during wound healing in these animals,
and this hypothesis is supported by the results described in this
study. We found a strong decrease in TGF-
1 and
2 mRNA
expression by dexamethasone in normal and wounded skin. Since these
factors have been shown to play an important role in granulation tissue
formation(14, 42) , our findings provide a likely
explanation for the delay of this process in glucocorticoid-treated
mice.
The molecular mechanisms that underlie the aberrant expression
of TGF-s during wound healing in glucocorticoid-treated mice are
currently unknown. However, our experiments suggest that a combination
of direct and indirect mechanisms might be responsible. Thus,
inhibition of inflammatory cell infiltration and activation by
glucocorticoids might indirectly reduce expression of TGF-
in the
wound, since activated macrophages and neutrophils are an important
source of TGF-
s, particularly of
TGF-
1(4, 5) . However, a direct effect of
glucocorticoids on other TGF-
-producing cells in the wound cannot
be excluded. Our finding that TGF-
1 and
2 expression is also
reduced by dexamethasone treatment in nonwounded skin supports this
hypothesis, since macrophages are present at low levels in normal skin.
Furthermore, preliminary experiments from our laboratory suggest a
negative regulation of TGF-
2 expression in keratinocytes. (
)Since these cells are an important source of TGF-
s in
the wound (8, this study), inhibition of TGF-
2 expression in these
cells by dexamethasone might also contribute to the reduced expression
of this gene in glucocorticoid-treated mice. Furthermore, decreased
expression of TGF-
1 and increased expression of TGF-
3 in the
wounds of dexamethasone-treated mice might be due to a direct effect of
glucocorticoids on mesenchymal cells. Thus, TGF-
1 mRNA and protein
expression have been shown to be reduced by glucocorticoids in lung
fibroblasts(43) , and TGF-
3 was recently identified as a
glucocorticoid-induced gene in the same cell type(44) .
In
summary, our data demonstrate a strong up-regulation of TGF- and
TGF-
receptor expression after injury which is modulated in a
complex manner by glucocorticoids. Since these steroids are potent
inhibitors of the wound healing process, our findings suggest that a
correct regulation of TGF-
and TGF-
receptor expression is
important for normal wound repair. Most importantly, these findings
provide a molecular explanation for the beneficial effects of high
concentrations of exogenous TGF-
on wound healing in
glucocorticoid-treated animals.