1 I. Medical Department, Section Pathophysiology, Johannes Gutenberg-University,
D-55131 Mainz, Germany
2 Institute of Pathology, University of Cologne, D-50931 Cologne, Germany
* Author for correspondence (e-mail: blessing{at}mail.uni-mainz.de )
Accepted 7 February 2002
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Summary |
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A cDNA array identified the transcription factor early growth response factor 1 (Egr1) as a target gene for TGFß in late phases of the wound healing process. As a member of the immediate-early gene family, Egr1 is upregulated shortly after injury and induces the expression of growth factor genes. We could demonstrate that Egr1 expression is also upregulated in skin wounds which have already undergone re-epithelialization. In conclusion, we attribute the enhanced re-epithelialization in our transgenics to the resistance of keratinocytes to TGFß-mediated growth restriction and apoptosis induction. We also propose a new role for TGFß induced Egr1 in late phase wound repair.
Key words: TGFß, Egr1, Wound healing, Re-epithelialization, Apoptosis, Mouse
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Introduction |
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A recently published wound healing study using Smad3 null mice
(Ashcroft et al., 1999) has
shed some light on this paradox. Re-epithelialization in these mice, which
lack a component of the TGFß cascade, was accelerated. The elevated BrdU
labeling index of keratinocytes at the wound edge demonstrated that TGFß
signaling can indeed inhibit keratinocyte proliferation in vivo and thereby
inhibit re-epithelialization. Nevertheless, owing to the fact that in this
knockout mouse the TGFß signaling pathway via Smad3 is abrogated in all
cells but may take place via Smad2, it is difficult to distinguish primary and
secondary effects on keratinocytes
(Ashcroft et al., 1999
). To
circumvent this problem, we have investigated wound healing of full thickness
excisional wounds in a mouse model with an interrupted TGFß signaling
pathway specifically in keratinocytes. We and others have previously shown
that the expression of a dominant negative type II TGFß receptor in
keratinocytes impairs the TGFß signal-transduction pathway in the
epidermis (Amendt et al., 1998
;
Wang et al., 1997
). Owing to
the use of the bovine keratin 5 promoter, the signal transduction pathway is
disrupted in keratinocytes with proliferative potential. This defined
interruption of TGFß signaling gives us the unique opportunity to
differentiate between direct and secondary effects of TGFß on
keratinocytes.
In this study, we have confirmed that TGFß impairs
re-epithelialization by inhibiting keratinocyte proliferation. In addition, we
could demonstrate that resistance to TGFß reduces keratinocyte apoptosis.
Furthermore, we demonstrate that the expression of the transcription factor,
early growth response gene 1 (Egr1) is upregulated in late phases of wound
healing. Moreover, we could show that this raised expression of Egr1 in late
phase wounds is induced by TGFß. Egr1 is a member of the immediate-early
gene family and has been shown to be induced shortly after injury
(Khachigian et al., 1996;
Pawar et al., 1995
). Egr1,
also known as NGFI-A, zif268, tis8 and Krox24, is the prototype member of the
early growth response gene family (Egr1-Egr3) (for reviews, see
Gashler and Sukhatme, 1995
).
Members of this family are rapidly induced by a variety of extracellular
stimuli including growth factors and cytokines, hypoxia, physical forces and
injury (Khachigian et al.,
1996
; Lemaire et al.,
1988
; Schwachtgen et al.,
1998
; Yan et al.,
1999
). During injury, Egr1 is considered to be a major
transcription factor for genes encoding crucial cytokines and growth factors
in injury repair, such as interleukin 2 (IL2), tumor necrosis factor
(TNF
), platelet-derived growth factors A and B (PDGF-A and PDGF-B),
basic fibroblast growth factor (bFGF) and TGFß
(Biesiada et al., 1996
;
Khachigian et al., 1996
;
Liu et al., 1996
;
Skerka et al., 1995
;
Yao et al., 1997
). Our finding
that Egr1 is upregulated by TGFß in keratinocytes at late stages of the
wound healing process provides new insights into the specific function of
TGFß signaling in keratinocytes: the induction of master regulators for
essential factors of tissue repair and remodeling.
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Materials and Methods |
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BrdU-labeling and apoptosis
BrdU-labeling experiments were performed using the In Situ Cell
Proliferation Kit, AP (Boehringer Mannheim, Mannheim, Germany) essentially as
described (Amendt et al.,
1998). In brief, mice were injected with 30 µg of BrdU per gram
body weight and sacrificed after a labeling period of 1 hour. Fixation and
processing of the samples were performed according to the manufacturers
instructions. BrdU indices were determined by photographing sections and
counting labeled nuclei in a 64 mm2 field. Detection and
quantification of programmed cell death in the epidermis was performed using
the In Situ Cell Death Detection Kit, FLUOS (Boehringer Mannheim,
Mannheim, Germany). Fresh cryosections were treated as recommended by the
supplier, counterstained for keratin 14, and analyzed by fluorescence
microscopy, essentially as described
(Breuhahn et al., 2000
). The
number of apoptotic cells in the basal epidermal layer was determined and
related to 100 total basal cells. Nuclei were stained with DAPI
(4',6-diamidino-2-phenylindole; Boehringer Mannheim, Mannheim, Germany).
Numbers given for analysis of apoptosis are mean values obtained from at least
four different animals and numbers given for BrdU-labeling experiments are
mean values obtained from at least three different animals.
Histology and immunohistology
Cryosections were cut at 5 µm thickness and fixed in 4% paraformaldehyde
in neutral buffered saline for 20 minutes at room temperature. Neutrophils and
mast cells were detected by histochemical staining of the sections with
naphthol-ASD-chloroacetate-esterase. Treatment of samples for
immunohistological procedures has been described previously
(Breuhahn et al., 2000). In
this study, an antibody directed to Mac-1 (CD11b, 1:100; Pharmingen, Germany,
Catalog No. 01711D) was used for the detection of macrophages.
Immunolocalization of Egr1 was carried out by using anti-mouse anti-Egr1
antibody (C-19, 1:100, Santa Cruz Biotechnology, Catalog no. sc-19)
(Ghanem et al., 2000
).
Incubation with the primary antibodies was performed overnight at 4°C.
RNA isolation and northern blot
Total RNA was isolated using Tri Reagent (Sigma-Aldrich Chemie GmbH,
Taufkirchen, Germany). Aliquots (30 µg) of RNA were electrophoresed on
1% agarose formaldehyde gels and subsequently blotted onto nylon membranes
(Hybond N, Amersham, Braunschweig, Germany). Filters were processed at high
stringency as described (Church and
Gilbert, 1984
). For analysis of wound specimens, RNA obtained from
three different animals was pooled. Probes used were either cDNA-fragments or
generated by RT-PCR with the following primers.
GAPDH: GAPDH-1, 5'-CAA CTA CAT GGT CTA CAT GTT C-3' (position 159-181; GenBank Accession Number M32599); and GAPDH-2, 5'-ACC AGT AGA CTC CAC GAC-3' (position 340-322; GenBank Accession Number M32599)
Egr1: EGR-1, 5'- AGC ACC TGA CCA CAG AGT CC-3' (position 578-597); and EGR-2, 5'- AGG TCT CCC TGT TGT TGT GG-3' (position 1078-1059; GenBank Accession Number NM_007913)
cDNA probes were used for detection of the human dominant negative type II TGFß receptor (human TGFß type II receptor, Accession Number NM_003242) and for keratin 5 (bovine cytokeratin III, Accession Number K03536). Expression levels were quantified using a PhosphorImager System (Molecular Dynamics STORM 860 System) and the Image Quant Software (Molecular Dynamics, Sunnyvale, USA). The band intensities of different mRNA species were related to the band intensities of the housekeeping gene Gapdh.
Genomic expression arrays
cDNA array analysis was performed by using AtlasTM Mouse 1.2 Arrays
(CLONTECH, Heidelberg, Germany) that contain a total of 1176 cDNA segments
spotted on a nylon membrane. Probing of cDNA arrays was performed as described
in the CLONTECH Atlas cDNA Expression Arrays User Manual (PT3140-1). Briefly,
RNA was extracted from 7-day-old wounds of three different animals using the
AtlasTM Pure Total RNA Labeling System according to the User Manual
(PT3231-1). The RNA was pooled and radiolabeled with
-p32-dATP from Amersham. After hybridization, the array
membrane was washed and analyzed by using PhosphorImager System (Molecular
Dynamics, Sunnyvale, USA). For normalization, cDNAs that did not show variable
intensities and were located near the cDNA of interest were used.
Cell culture and transfection of HaCaT cells
HaCaT cells (Boukamp et al.,
1988) were maintained in GC-Medium supplemented with 0.5% FCS
(Vitromex, Weirdo, Germany). Stable DNA transfections were carried out using
the calcium phosphate procedure (Graham
and van der Eb, 1973
). The expression vector pHbAPr-1 consists of
a 4.3 kb fragment of the human ß-actin gene promoter, a polylinker
derived from pSP64 and the vector backbone derived from pcDV1, including the
AmpR gene and NeoR gene (Leavitt et al.,
1984
; Melton et al.,
1984
; Okayama and Berg,
1983
). The cDNA of the dominant negative type II receptor was
cloned into the polylinker (Brand et al.,
1993
). The transfectants were selected in medium containing 1
mg/ml G418. For RNA-preparation, cells were lysed directly in Tri Reagent 45
minutes after addition of 5 ng/ml TGFß1 (Strathmann Biotec AG, Hamburg,
Germany).
Statistics
Data is shown as mean±s.d. Statistical significance in the
differences between two groups was analyzed using Student's t-test
and values of P<0.05 were considered significant.
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Results |
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|
|
Remodeling
The accelerated re-epithelialization process in the transgenic mice
correlated with an earlier loss of hematopoietic cells from the wound site
(Fig. 2). Sections of wound
specimens from at least four different animals were Giemsa stained,
photographed and the number of hematopoietic cells were quantified in a 64
mm2 field (Fig. 2).
Ten days after wounding, no significant difference in the numbers of
hematopoietic cells was evident (Fig.
2E). But at day 14, the number of hematopoietic cells in the wound
was markedly decreased in the transgenic animals compared with the control
animals (Fig. 2E; 1.6-fold
reduction, P<0.02). To further clarify which blood-derived cells
were retained in the wounds of non-transgenic animals, we performed
immunohistochemical staining for macrophages, using the macrophage-specific
marker Mac1, and for mast cells and neutrophils, using
naphthol-ASD-chloroacetate-esterase staining, in two transgenic and
non-transgenic animals (Fig.
3). In 14-day old wounds of transgenic animals we found fewer
macrophages and mast cells or neutrophils
(Fig. 3A,C) when compared with
non-transgenic animals (Fig.
3B,D). The earlier disappearance of the hematopoietic cells in the
wound site of the transgenic animals correlates with the accelerated wound
closure.
|
|
Keratinocyte proliferation and apoptosis
To test if the interrupted TGFß signaling pathway in keratinocytes
leads to a relaxed cell cycle control and thus to a higher proliferation rate
in the wound edge of transgenic mice, we performed BrdU-labeling experiments
with at least three different animals per group
(Fig. 4). At days 3, 5 and 7,
mice of the transgenic line 114 showed significantly increased numbers
(P<0.05, Student's t-test) of labeled nuclei in the wound
edge compared with non-transgenic animals
(Table 2). Similarly,
transgenic mice of line 54, which shows a lower level of transgene expression
than line 114, also displayed elevated BrdU labeling indices in comparison
with controls, albeit these differences were less pronounced than in line 114
(Table 2). This finding
correlates well with the different levels of transgene expression in these two
lines (Amendt et al., 1998).
These results demonstrate that the accelerated re-epithelialization in
transgenic mice could be attributed at least in part to an increased
proliferation rate in keratinocytes at the wound edge.
|
|
It had been shown that the decrease of cellularity in the remodeling phase
during wound healing involves apoptosis
(Desmouliere et al., 1995). As
TGFß induces programmed cell death in various cell types
(Lomo et al., 1995
;
Nass et al., 1996
), we tested
if apoptosis was modulated in keratinocytes during the late phase of wound
healing. Apoptotic cells in 13-day-old wounds were marked by TUNEL labeling
and counted versus total number of basal cells in at least four different
transgenic and non-transgenic animals (Fig.
5). In transgenic animals, a significantly lower number of
apoptotic cells were found in comparison with wild-type animals. Only
0.42±0.16% basal epidermal cells in transgenic animals undergo
apoptosis, whereas in wild-type animals 0.77±0.10% of the basal
epidermal cells were apoptotic (P<0.005, Student's
t-test). RNAse protection assays using RNA from pooled wounds
obtained from three different animals at 3 days and 5 days after wounding
showed that the Bcl2 family members Bcl2, Bcl-X, Bax, Bak, Bad and A1 were not
differentially regulated on the RNA level between transgenic and wild-type
animals (data not shown).
|
Target genes
To find TGFß target genes in keratinocytes during wound healing, we
performed a cDNA array hybridization. cDNA from pooled wounds of at least
three transgenic and non-transgenic animals collected at day 7 post wounding
were used as probes. We found that the gene encoding early growth response
factor 1, a transcription factor, was markedly downregulated (sevenfold) in
transgenic animals compared with non-transgenic animals
(Fig. 6A). We confirmed the
finding that Egr1 is upregulated in controls even at this late phase of the
wound healing process using northern blot analysis of 13-day-old wounds. For
this analysis pooled RNA from three different animals were used. In unwounded
skin, the expression of Egr1 mRNA was low in both transgenic and non
transgenic animals. Whereas in 13-day-old wounds, the expression in
non-transgenic animals was 4.8-fold (mean of three independent experiments)
higher compared with unwounded skin. By contrast, in 13-day-old wounds of
transgenic animals, only a 1.5-fold upregulation (P<0.05,
Student's t-test) of Egr1 expression was detected
(Fig. 6B,C show one
representative experiment).
|
The cells mainly accounting for Egr1 expression in response to wounding are keratinocytes, as demonstrated by Egr1 immunostaining in 13-day-old wounds (Fig. 7). Keratinocytes of the newly re-epithelialized epidermis showed strong Egr1 staining, mainly in the cytoplasm (Fig. 7A). By contrast, in unwounded areas, Egr1 expression in keratinocytes was not detectable (Fig. 7B).
|
For further confirmation of an impaired upregulation of Egr1 in keratinocytes by an interrupted TGFß signaling pathway, we stably transfected the human keratinocyte line HaCaT with an expression vector containing the dominant negative type II TGFß receptor (Fig. 8). The expression of the dominant negative type II TGFß receptor in two transfected HaCaT clones was analyzed by Northern Blot analysis (Fig. 8A). Clone 1 expressed the dominant negative type II TGFß receptor at high levels, in contrast to clone 2, which is used as vector control (neo). The induction of Egr1 mRNA in response to TGFß was measured by northern blot analysis and subsequent quantitative evaluation was performed using a PhosphorImager system (Fig. 8B,C). In wild-type HaCaT cells and in the transfected control cells (neo), the expression of Egr1 mRNA was strongly induced by TGFß1 (25-fold). By contrast, HaCaT cells, which express the dominant negative type II TGFß receptor at high levels showed a markedly reduced upregulation of Egr1 mRNA (ninefold, n=3, P=0.0005, Student's t-test). This confirms the assumption that the dominant negative type II TGFß receptor is able to abolish or reduce induction of Egr1 expression in keratinocytes.
|
![]() |
Discussion |
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Members of the immediate-early gene family, which are expressed instantly
after injury, regulate and orchestrate the tissue repair process
(Khachigian et al., 1996;
Liu et al., 2000b
;
Wang and Deuel, 1992
). Because
of this, studies investigating the role of Egr1, a member of this
immediate-early gene family, during injury have focused on monitoring the
expression of Egr1 for only a short period after wounding
(Bryant et al., 2000
;
Khachigian et al., 1996
). We
demonstrate that Egr1 upregulation in wounds is predominantly seen in
keratinocytes. The cytoplasmic pattern of staining correlates with
observations from kidney epithelial cells from Wilms tumors
(Ghanem et al., 2000
).
Using a cDNA array and northern blot analysis, we found an elevated
expression level of Egr1 in late phase wounds of wild-type animals. This novel
finding of late-stage Egr1 expression during wound healing is of significance
in consideration of the observed functions of Egr1 in developmental and
differentiation processes (Milbrandt,
1987; Sukhatme et al.,
1988
). Moreover, we could demonstrate that this late upregulation
of Egr1 during wound healing is caused by TGFß, which is upregulated
during wound repair with peak levels as early as 1-2 days post-wounding
(Frank et al., 1996
).
Transgenic animals with an interrupted TGFß signaling cascade show no
upregulation of Egr1 expression. This TGFß-dependent Egr1 upregulation in
keratinocytes is further confirmed by our finding that interruption of the
TGFß signal pathway in the human keratinocyte cell line HaCaT also leads
to impaired upregulation of Egr1. Hence, there is the possibility that Egr1
expression is regulated by an autocrine loop. On the one side, Egr1 is induced
by TGFß in various cell lines such as the osteoblastic cell line MC3T3,
the fibroblast cell line NIH 3T3 and the epithelial cell line NMuMG
(Koskinen et al., 1991
;
Ohba et al., 1994
). On the
other side, Egr-1 induces TGFß1 by binding to GC-rich binding sites in
the promotor region of TGFß1 (Kim et
al., 1994
; Liu et al.,
1996
).
Two recently published wound-healing studies have demonstrated the
importance of Egr1 in early wound healing. The delivery of the cDNA for Egr1
by a gene gun in a full excisional wound model in mice leads to accelerated
wound healing, owing to induction of cytokines such as vascular endothelial
growth factor (VEGF), PDGF-A and TGFß1
(Bryant et al., 2000). A
DNA-based enzyme that degrades Egr1 mRNA resulted in impaired wound healing of
arterial neointima injured by a balloon catheter
(Santiago et al., 1999
). In
contrast to the function of Egr1 in the immediate response to injury, very
little is known about the function of Egr1 expression in late phases of wound
repair. But consistent with its role in developmental and differentiation
processes, as well as in the control of cell growth, Egr1 is very probably an
important factor for the remodeling and termination phase of the wound healing
process (Dinkel et al., 1998
;
Krishnaraju et al., 2001
;
McMahon et al., 1990
;
Santiago et al., 1999
). The
finding that Egr1 is expressed more continuously in developmental and
differentiation processes favors this hypothesis
(Milbrandt, 1987
;
Sukhatme et al., 1988
).
Additional support for this assumption is the finding that in chronic wounds
like atheriotic lesions, levels of Egr1 expression remain elevated. In these
lesions, Egr1 seems to repress the transcription of the type II TGFß
receptor and thus contributes to acquired resistance of the lesional cells to
effects of TGFß (Du et al.,
2000
; McCaffrey et al.,
2000
). Furthermore, Egr1 is able to induce the expression of
fibroncetin in cell lines derived from a glioblastoma and a fibrosarcoma and
of metalloproteinases in endothelial cells
(Haas et al., 1999
;
Liu et al., 1999
;
Liu et al., 2000a
). This
indicates that Egr1 is capable of transactivating genes relevant for later
phases of wound repair.
Resolving the wound repair process is another crucial step in wound healing
that involves apoptosis in order to decrease cellularity
(Desmouliere et al., 1995).
Egr1 and other members of the immediate-early gene family have been linked to
apoptosis induction whereby Myc, Fos and Egr1 mediate the proapaptotic signal
via p53 (Estus et al., 1994
;
Hermeking and Eick, 1994
;
Liu et al., 2001
;
Muthukkumar et al., 1995
;
Nair et al., 1997
;
Preston et al., 1996
;
Woronicz et al., 1994
). The
reduced Egr1 expression found in 13-day-old wounds in transgenic animals
correlated with a reduced rate of apoptosis in the epidermis. This suggests
that TGFß-induced Egr1 also plays a role in the resolution phase of wound
repair by inducing apoptosis in keratinocytes.
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Acknowledgments |
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