©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factors 1, 2, and 3 and Their Receptors Are Differentially Regulated during Normal and Impaired Wound Healing (*)

(Received for publication, October 30, 1995; and in revised form, February 9, 1996)

Stefan Frank (§) Marianne Madlener Sabine Werner (¶)

From the Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A series of studies has shown that application of transforming growth factor beta (TGF-beta) to a wound has a beneficial effect, especially in animals with wound healing disorders. In this study we have investigated the regulation of TGF-beta1, beta2, and beta3 and their receptors during the repair process. We found a large induction of all three TGF-beta isoforms and also of TGF-beta types I and II receptors, although the time course of induction and the absolute expression levels were different for these genes. Furthermore, each TGF-beta isoform had distinct sites of expression in the wound. Systemic treatment with glucocorticoids significantly altered the expression levels of TGF-betas and TGF-beta receptors. Whereas expression of TGF-beta1, TGF-beta2, and TGF-beta type II receptor was suppressed by glucocorticoids in normal and wounded skin, expression of TGF-beta3 and TGF-beta receptor type I mRNA was stimulated. These findings provide an explanation for the beneficial effect of exogenous TGF-beta 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-beta isoforms and their receptors might underlie the wound healing defect seen in glucocorticoid-treated animals.


INTRODUCTION

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 beta (TGF-beta), (^1)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-beta isoforms (TGF-beta1, beta2, and beta3) are present in mammals that share a 64-85% amino acid sequence homology(10) . In vitro, TGF-betas 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-betas 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-beta are expressed during wound repair, whereby each member of the TGF-beta family has a unique distribution in pig wounds(8) . The differential expression of TGF-betas 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-beta1, beta2, and beta3 have been demonstrated. These include the inhibitory effect on DNA synthesis in keratinocytes, where TGF-beta3 is significantly more potent than TGF-beta1 and TGF-beta2(13) . Recently Shah et al.(14) were able to demonstrate differences in the biological effects of TGF-betas during wound repair. Their studies suggest that TGF-beta1 and TGF-beta2 induce cutaneous scarring, whereas TGF-beta3 seems to inhibit this effect.

Whereas increased expression of TGF-beta1 and TGF-beta2 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-beta had favorable effects on the repair process(17, 18, 19, 20) . Therefore, we speculated that expression of endogenous TGF-betas might also be impaired in these animals. Here we demonstrate severe effects of glucocorticoids on the expression of TGF-betas 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.


MATERIALS AND METHODS

Glucocorticoid Treatment of Mice

Female BALB/c mice (3 months old) were injected subcutaneously at 9 a.m. daily with 1 mg of dexamethasone/kg of body weight for 7 days. Control mice were injected with phosphate-buffered saline. Three dexamethasone-treated mice and three control mice were subsequently injected with dexamethasone or phosphate-buffered saline for another 3 days but left unwounded. The other mice were wounded as described below. During the wound healing period, we continued the daily injection of dexamethasone or phosphate-buffered saline.

Wounding and Preparation of Wound Tissues

To assess TGF-beta and TGF-beta receptor expression during wound healing, six full-thickness wounds were created on each animal, and skin biopsy specimens from four animals were obtained 1, 3, 5, 7, and 13 days after injury. To study the effect of dexamethasone on TGF-beta expression, wounds from four dexamethasone-treated mice and four control mice were analyzed at day 2, 3, or 5 after injury. Mice were anesthetized with a single intraperitoneal injection of Avertin. The hair on the back of these mice was cut with fine scissors, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (6-mm diameter, 3-4 mm apart) were made on the backs of these mice by excising the skin and panniculus carnosus. The wounds were allowed to dry to form a scab. An area of 7-8 mm in diameter which included the scab and the complete epithelial margins was excised at each time point. A similar amount of skin from the backs of three nonwounded animals was used as a control. In every experiment, the wounds from four animals (24 wounds) and the nonwounded back skin from three animals, respectively were combined, immediately frozen in liquid nitrogen, and stored at -70 °C until used for RNA isolation. In one experiment, four additional mice (two control mice and two glucocorticoid-treated mice) were wounded. The wounds of these mice were analyzed histologically at day 5 after injury to study the effects of glucocorticoid treatment on wound healing. All animal experiments were carried out with permission from the local government of Bavaria (permission number 211-2531-16/93).

Histology

Glucocorticoid-treated mice and control mice were wounded as described above. Animals were sacrificed at day 5 after injury. Complete wounds were isolated from the middle of the back, bisected, fixed overnight in 4% paraformaldehyde, and paraffin embedded. Six-µm sections from the middle of the wound were stained with hematoxylin and eosin.

Preparation of Tissue Lysate and Enzyme-linked Immunosorbent Assay

Normal and wounded back skin was frozen in liquid nitrogen. Eight-cm^2 of normal and wounded back skin (10 wounds) were homogenized in 3 ml of 2 times lysis buffer (1 times lysis buffer: 1% Triton X-100, 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin (0.15 unit/ml), 15 µg/ml leupeptin, 10 µg/ml pepstatin). The tissue extract was cleared by centrifugation and the supernatant diluted 1:1 with water. One ml of each diluted sample was cleared by centrifugation. The amount of protein in the lysate was determined using the Bio-Rad protein assay (Bradford method). One µg of total protein from skin and wound lysates was treated with 1 N HCl to activate latent TGF-beta. 65 and 130 ng of HCl-treated protein lysate were subsequently analyzed for the presence of immunoreactive TGF-beta1 by enzyme-linked immunosorbent assay using the Predicta TGF-beta1 kit (Genzyme) as described by the manufacturer.

Immunohistochemistry

Mice were wounded as described above. Animals were sacrificed at day 5 after injury. Complete wounds were isolated from the middle of the back, bisected, and frozen in OCT. Six-µm frozen sections were fixed with acetone and treated for 10 min at room temperature with 1% H(2)O(2) in phosphate-buffered saline to block endogenous peroxidase activity. They were subsequently incubated for 60 min at room temperature with polyclonal antisera for TGF-beta1, beta2, or beta3 (Santa Cruz, Biotechnologies) (1:250 diluted in phosphate-buffered saline, 0.1% bovine serum albumin). These antibodies have been shown to be specific for the different TGF-beta isoforms as assessed by Western blot analysis and immunohistochemical staining. The slides were subsequently stained with the avidin-biotin-peroxidase complex system (Vector Laboratories, Burlingame, CA) using 3-amino-9-ethylcarbazole as a chromogenic substrate. After development, they were rinsed with water, counterstained with hematoxylin, and mounted.

RNA Isolation and RNase Protection Assay

RNA isolation was performed as described(21) . Fifty µg of total RNA from wounded or nonwounded skin was used for RNase protection assays. RNase protection assays were carried out as described recently(22) . Briefly, DNA probes were cloned into the transcription vector pBluescript KSII (+) (Stratagene) and linearized. An antisense transcript was synthesized in vitro using T3 or T7 RNA polymerase and [P]UTP (800 Ci/mmol). RNA samples were hybridized at 42 °C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested with RNases A and T1 for 1 h at 30 °C. Under these conditions, every single mismatch is recognized by the RNases. Thus, cross-reaction of the individual probes with other TGF-beta isoforms can be excluded. Protected fragments were separated on 5% acrylamide, 8 M urea gels and analyzed by autoradiography. All protection assays were carried out with at least two different sets of RNA from independent wound healing experiments.

Probe DNAs

Human and murine cDNA probes for TGF-beta1, beta2, and beta3 were cloned by polymerase chain reaction using 5`-GA(A/G)TGG(C/T)TI(A/T)(C/G)ITT(C/T)GA(C/T)GT-3` as a 5`-primer and 5`-GG(C/T)TC(A/G)TG(A/C/G/T)A(C/T)CCA(C/T)TTCCA-3` as a 3`-primer. These primers correspond to regions that are highly conserved among the different forms of TGF-beta; therefore, they could be used for the amplification of the three TGF-beta cDNAs. The murine TGF-beta type I receptor (Alk-5) cDNA was cloned by polymerase chain reaction using 5`-ATCCATGAAGACTATCAGTTGCCT-3` as a 5`-primer and 5`-CATTTTGATGCCTTCCTGTTGGCT-3` as a 3`-primer. The amplified fragment corresponds to nucleotides 1258-1509 of the published sequence(23, 24) . The murine TGF-beta type II receptor cDNA probe was cloned by polymerase chain reaction using degenerated primers corresponding to the human cDNA (25) (5`-primer: 5`-GGNGARACNTTYTTYATGTG-3`; 3`-primer: 5`-TTDATRTTRTTNGCRCANGT-3`).


RESULTS

Expression of TGF-betas in the Dermis and the Epidermis

To determine potential sites of action of TGF-betas in normal skin, we first investigated the endogenous expression of TGF-beta1, TGF-beta2, and TGF-beta3 as well as their receptors in the dermis and epidermis of mouse skin. Tail skin was used for this purpose, since this is the only place where the dermis can be separated from the epidermis. For detection of the individual transcripts, RNase protection assays were performed under conditions where a single base mismatch can be detected. Thus, cross-reaction of the probes with other TGF-beta isoforms could be excluded. This was further proven by hybridization of the probes with TGF-beta1, beta2, and beta3 sense transcripts (data not shown). We found expression of TGF-beta1 and TGF-beta2 in the dermis and at lower levels in the epidermis (Fig. 1, A and B). By contrast, TGF-beta3 mRNA could only be detected in the dermis, although at much lower levels compared with the other types of TGF-beta (Fig. 1C). TGF-beta signals by contacting two distantly related transmembrane serine/threonine kinases called receptors I and II(25, 26, 27) . As shown in Fig. 1, D and E, both receptors were expressed in mouse tail skin. mRNA encoding the type I receptor (TGF-betaRI) was found at similar levels in the dermis and the epidermis (Fig. 1D). The type II receptor (TGF-betaRII) was found at high levels in the dermis, whereas epidermal expression was low and could only be detected after longer exposure times (Fig. 1E and data not shown).


Figure 1: Expression of TGF-betas and TGF-beta 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-betas and TGF-beta 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.



Expression of TGF-beta1, TGF-beta2, and TGF-beta3 Is Differentially Regulated during Wound Repair

To investigate the mRNA expression of TGF-betas in normal and wounded skin, we isolated RNA from full-thickness wounds at different intervals after injury and performed RNase protection assays. For each time point, 24 wounds from four mice were excised, combined, and used for RNA isolation. Normal skin from the back of nonwounded mice was used as a control. As shown in Fig. 2, A-C, TGF-beta1, beta2, and beta3 were expressed in normal back skin. Upon injury, a strikingly increased expression of all TGF-beta isoforms was observed, although the kinetics of induction was different for the three variants. Expression of TGF-beta1 mRNA increased 9-fold within 24 h after wounding and remained high for several days after injury (Fig. 2, A and D). Expression of TGF-beta3 mRNA was low during the first 3 days after injury. However, TGF-beta3 mRNA levels subsequently increased, and a maximal induction (12.5-fold) was observed at day 7 after wounding (Fig. 2, C and D). Thus, TGF-beta3 expression levels were highest during the period of maximal granulation tissue formation and reepithelialization. Similar kinetics were observed for TGF-beta2, although induction of this gene was only 4.5-fold (Fig. 2, B and D). At day 13 after injury all wounds were completely reepithelialized, but the granulation tissue still revealed a high cellularity (data not shown). At that stage of the repair process, expression of all TGF-beta variants had declined, although the expression levels were still significantly higher compared with the basal level (Fig. 2D). This suggests that TGF-betas are also involved in the late stage of the repair process which is characterized by significant tissue remodeling. This time course of TGF-beta1, beta2, and beta3 expression was seen in three different RNase protection assays with RNAs from independent wound healing experiments.


Figure 2: Differential expression of TGF-beta1, TGF-beta2, and TGF-beta3 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-beta1 (panel A), TGF-beta2 (panel B), and TGF-beta3 (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-beta1, beta2, and beta3 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-beta1, beta2, and beta3 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-beta1, beta2, and beta3 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-beta 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-beta cDNAs. These results revealed a similar expression of TGF-beta1 and beta3 at later stages of the repair process (5-7 days after injury), whereas TGF-beta2 mRNA levels were 90% lower (data not shown). Since TGF-beta3 expression is significantly lower during the early phase of wound healing (days 1-3 after injury) (Fig. 2), TGF-beta1 seems to be the predominant isoform during this period.

Induction of TGF-beta mRNA Expression after Injury Correlates with Induction of Immunoreactive TGF-beta Protein

To determine whether the observed induction of TGF-beta expression after injury correlates with induction of TGF-beta protein, we determined the amounts of immunoreactive TGF-beta1 protein in normal and wounded skin by enzyme-linked immunosorbent assay. For this purpose, tissue lysates were prepared from normal and wounded skin (2 days after injury), and 65 or 130 ng of total protein was analyzed for the presence of TGF-beta1. Lysate from normal skin contained 0.1 ng/ml immunoreactive TGF-beta1, whereas the levels of this factor were 4-fold higher (0.43 ng/ml) in the lysate of wounded skin. This result demonstrates that induction of TGF-beta1 mRNA correlates with increased production of TGF-beta1 protein.

To determine the localization of the different types of TGF-beta in normal and wounded skin, serial sections from 5-day full-thickness mouse wounds were stained with monospecific antibodies against TGF-beta1, beta2, and beta3. At that time point after injury, all types of TGF-beta are expressed at high levels ( Fig. 2and Fig. 3). As shown in Fig. 3, A-C, TGF-beta1, beta2, and beta3 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-beta2 protein was particularly abundant below the hyperproliferative epithelium (Fig. 3B). TGF-beta3 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-beta1 protein was seen in differentiating keratinocytes of the hyperthickened epidermis (Fig. 3A), and TGF-beta2 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-beta isoforms in the wound tissue.


Figure 3: Differential expression of TGF-beta1, beta2, and beta3 proteins in 5-day mouse wounds. Frozen serial sections from a 5-day mouse wound were incubated with monospecific antibodies directed against TGF-beta1 (panel A), TGF-beta2 (panel B), or TGF-beta3 (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.



Expression of TGF-betas during Wound Healing Correlates with Expression of TGF-beta Type I and Type II receptors

To determine whether expression of TGF-betas correlates with expression of the corresponding receptors, we analyzed the mRNA levels of both types of TGF-beta receptor at different stages of the repair process. As shown in Fig. 4, high levels of TGF-beta type II receptor mRNA but significantly lower levels of the type I receptor mRNA were found in normal murine back skin. A transient increase in expression of both receptors was seen after injury. Expression levels were maximal at day 1 after injury and subsequently declined. After completion of the proliferative phase of wound repair (13 days after injury) expression levels were still elevated compared with control skin. The presence of both types of receptors in normal and wounded skin and their up-regulation after injury demonstrate a strong correlation between expression of the ligands and their receptors.


Figure 4: TGF-beta 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-beta type I (TGF-betaRI) and type II (TGF-betaRII) 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.



Glucocorticoids Have Differential Effects on TGF-beta1, beta2, and beta3 Expression in Normal Skin and during Wound Healing

Inhibition of wound healing by glucocorticoids is a well established phenomenon. To determine a possible role of TGF-beta in impaired wound healing of glucocorticoid-treated mice, dexamethasone was injected daily at 9 a.m. over a period of 7 days before wounding and 2-5 days after wounding. To prove the deleterious effect of dexamethasone treatment on wound repair, wounds were analyzed histologically at day 5 after injury. A significant amount of granulation tissue had formed in control mice at this time point, whereas in dexamethasone-treated mice granulation tissue formation was hardly detectable. Furthermore, reepithelialization was dramatically delayed in glucocorticoid-treated mice (data not shown). RNA was isolated from nonwounded skin and from wounded skin of glucocorticoid-treated mice and control mice at day 2, 3, or 5 after injury and analyzed for TGF-beta mRNA expression.

In nonwounded skin, expression levels of TGF-beta1 and TGF-beta2 were reduced by dexamethasone (Fig. 5, A and B), whereas TGF-beta3 mRNA levels increased upon glucocorticoid treatment (Fig. 5C). These differences were also seen after injury, and expression levels of TGF-beta1 and TGF-beta2 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-beta1 and TGF-beta2, expression of TGF-beta3 mRNA was stimulated by dexamethasone, although this difference was only seen in the early phase of wound repair when TGF-beta3 mRNA expression is normally very low (Fig. 6C). Thus, systemic treatment with glucocorticoids resulted in a premature onset of TGF-beta3 expression. These in vivo data demonstrate that glucocorticoids modulate the normal induction of TGF-beta1, beta2, and beta3 expression after cutaneous injury.


Figure 6: TGF-beta1, TGF-beta2, and TGF-beta3 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-beta1 (panel A), TGF-beta2 (panel B), and TGF-beta3 (panel C) expression. One thousand cpm of the hybridization probes was used as a size marker. The degree of TGF-beta1, beta2, or beta3 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.



Differential Modulation of TGF-beta Type I and II Receptors by Glucocorticoids

In addition to its effects on TGF-beta expression, dexamethasone also modulated the expression levels of both TGF-beta receptors. In nonwounded skin, glucocorticoids had no effect on TGF-beta type I receptor expression (Fig. 7A), whereas TGF-beta type II receptor expression was significantly reduced upon dexamethasone treatment (Fig. 7B). During wound healing, glucocorticoid treatment resulted in increased expression of the type I receptor (Fig. 7A), whereas type II receptor mRNA levels were slightly reduced in the wounds of dexamethasone-treated mice (Fig. 7B). This finding demonstrates a differential regulation of the two TGF-beta receptors, suggesting that the responsiveness to TGF-beta might be altered upon glucocorticoid treatment.


Figure 7: Modulation of TGF-beta 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-beta type I and II receptors (TGF-betaRI and TGF-betaRII, respectively). One thousand cpm of the hybridization probes was used as a size marker.




DISCUSSION

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-beta. Expression of different TGF-beta 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-beta expression during the repair process. In this study we demonstrate a strong up-regulation of TGF-beta1, beta2, and beta3 expression after injury. These findings correlate with results from other authors who demonstrated increased TGF-beta-like activity in wound fluid from rats(31) .

The kinetics of expression was different for the three TGF-beta variants. Particularly remarkable was the strong induction of TGF-beta3 expression at later stages of the repair process. Since TGF-beta3 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-beta3 on connective tissue deposition. They demonstrated an increase in new dermal matrix by exogenous application of TGF-beta3 to wounds in age-impaired animal models(32) . However, the effect of TGF-beta3 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-betas are likely to be important in the epidermis. TGF-beta3 is the most abundant TGF-beta isoform in the hyperproliferative epithelium and might therefore play an important role in keratinocyte differentiation. Thus, increased expression of TGF-beta3 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-beta1, beta2, and beta3 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-beta2 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-beta3 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-beta2 and TGF-beta3, the expression pattern of TGF-beta1 in the epidermis was similar in all species, and TGF-beta1 protein was found at highest levels in the upper layers of the epidermis.

Recently, two different TGF-beta receptors have been cloned which mediate TGF-beta signal transduction. However, expression of these receptors in a wound has not yet been demonstrated. Here we show that both types of TGF-beta receptor are expressed at high levels in normal and wounded skin, suggesting that these receptors are indeed mediators of TGF-beta action in the wound. Interestingly, glucocorticoids had differential effects on the expression levels of both receptors, and the ratio of TGF-beta 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-betas 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-betas 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-beta 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-beta on wound healing in glucocorticoid-treated animals (17, 19, 20) suggested that endogenous TGF-beta 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-beta1 and beta2 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-betas 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-beta in the wound, since activated macrophages and neutrophils are an important source of TGF-betas, particularly of TGF-beta1(4, 5) . However, a direct effect of glucocorticoids on other TGF-beta-producing cells in the wound cannot be excluded. Our finding that TGF-beta1 and beta2 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-beta2 expression in keratinocytes. (^2)Since these cells are an important source of TGF-betas in the wound (8, this study), inhibition of TGF-beta2 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-beta1 and increased expression of TGF-beta3 in the wounds of dexamethasone-treated mice might be due to a direct effect of glucocorticoids on mesenchymal cells. Thus, TGF-beta1 mRNA and protein expression have been shown to be reduced by glucocorticoids in lung fibroblasts(43) , and TGF-beta3 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-beta and TGF-beta 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-beta and TGF-beta 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-beta on wound healing in glucocorticoid-treated animals.


FOOTNOTES

*
This work was supported in part by a grant from the Bundesministerium für Bildung und Forschung (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a postdoctoral fellowship from the Ernst Schering Research Foundation.

To whom correspondence should be addressed. Tel.: 089-8578-2269/2271; Fax: 089-8578-2814.

(^1)
The abbreviation used is: TGF-beta, transforming growth factor beta.

(^2)
M. Madlener, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Peter Hans Hofschneider for support and for helpful suggestions with the manuscript; Dr. Reinhard Fässler for helpful discussions; and Dr. Hans Smola, Universität Köln, for histological analysis of the wounds. We acknowledge gratefully the excellent technical assistance of Frederique Torterotot and Helga Riesemann.


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