The beneficial effect of exogenous growth factors in the treatment of wound repair as well as the identification of the in vitro activities of many growth factors and cytokines have implicated these proteins as key regulators of the wound healing process (Table 1). This hypothesis is strongly supported by the expression of multiple growth factors and their receptors in different cell types of healing skin wounds. In addition, upregulation of growth factor expression after injury is frequently observed, suggesting a need for high growth factor levels during the repair process. Finally, there are many examples where abnormal growth factor expression is associated with impaired wound healing or excessive scarring, indicating that a correct temporal and spatial expression of these genes is essential for normal repair.
To determine the in vivo function of endogenous growth factors and cytokines, several investigators have applied neutralizing antibodies to skin wounds or to wound fluid (Tables 2 and 3). In most cases, this treatment strongly affected the healing process and provided insight into the roles of growth factors in repair. However, these results have to be interpreted with care, since cross-reactivity with related growth factors cannot be excluded. Furthermore, it is unclear whether neutralizing antibodies get access to the complete wound area.
Given these limitations, a genetic approach to identify growth factor function in wound repair appears desirable. Indeed, the use of genetically modified mice for wound healing studies recently revealed crucial roles for several growth factors and cytokines in the repair process (Table 4). For example, these studies have demonstrated an inhibitory effect of TGF- on wound reepithelialization (2, 12, 66, 149), an important role of FGF receptor signaling in this process (292), and a role of activin in granulation tissue formation and scarring (192, 286). In the latter case, the phenotype was more pronounced in a CD1 background compared with a B6D2F2 background (Werner, unpublished data), demonstrating the importance of the genetic background for the outcome of the repair process.
In spite of the potency of the genetic approach for the study of gene function in wound repair, some of the normal functions of targeted genes might not be revealed due to redundancy or compensation. Indeed, lack of wound healing abnormalities was often observed in mice deficient in a particular growth factor that belongs to a multiprotein family, e.g., in mice lacking FGF7 or TGF- (113, 164, 171). In the latter case, however, a more detailed analysis of the healing process and the use of another wound model did finally reveal a function of TGF- in epithelial repair (146). Nevertheless, the observed phenotype was more subtle than expected from its known in vitro activities. Although it cannot be excluded that FGF7 and TGF- are indeed not important for wound repair, their strong induction in healing skin wounds supports their functional significance. In both cases other growth factors, which bind to the same receptor and which are also expressed in wounded skin, might compensate for the lack of these mitogens. Wound healing studies using animals, which are deficient in two or more homologous molecules, as well as studies with dominant-negative receptor mutants or with soluble inhibitors that block the function of several members of a protein family, will be very useful in answering these questions. For example, the overexpression of a dominant-negative FGFR2IIIb in the epidermis of transgenic mice demonstrated an important role of FGFR signaling in wound reepithelialization (292), whereas the knock-out of individual ligands did not reveal this function (113, 188, 208).
At the other extreme, systemic abnormalities caused by the transgene or by the general loss of a gene might obscure the normal function of a gene in wound repair. Thus it has long been known that systemic defects such as malnutrition and weight loss can strongly impair the healing process (108). This was observed for the TGF-1 knock-out mice, which developed a strong inflammatory response in various tissues and organs, followed by severe weight loss at 3 wk of age. These systemic defects appear to be responsible for the impaired wound healing seen in these animals (41), making it impossible to study the local effects of the lack of TGF-1 on wound repair in this model. Suitable approaches to circumvent this problem, as mentioned above, were to cross the mice onto an immunodeficient background or to treat them with immunosuppressive drugs (66, 149). These studies serve to alert us to the difficulties of determining the functions of specific proteins in complex in vivo situations, but provide some elegant methods for circumventing these problems.
Another way to solve this problem is the generation of mice that have a tissue-specific knock-out or tissue-specific overexpression of a transgene (218). For example, overexpression of a dominant-negative TGF- receptor in the epidermis of transgenic mice did not cause any systemic abnormalities, but nevertheless allowed the identification of an inhibitory effect of TGF- on wound reepithelialization (2). An even more elegant approach will be the use of inducible systems, which allow the induction of a transgene or the deletion of an endogenous gene in a time- and tissue-specific manner. The first successful results with inducible systems in the skin have recently been published. Two have adopted an estrogen receptor-based approach, where Cre recombinase was fused in frame with the tamoxifen-responsive hormone-binding domain of the estrogen receptor [CreER(tam)]. This fusion protein was expressed under the control of the keratin 5 (131) or keratin 14 promoters (280) that target transgenes to the basal layer of the epidermis and to the outer root sheath of hair follicles. Upon systemic or local application of tamoxifen, the latter binds to the hormone-binding domain of the fusion protein and causes activation of Cre recombinase. The activated enzyme then allows the deletion of the targeted gene in keratinocytes.
In an analogous approach, activation of other intracellular proteins can be achieved by fusing the cDNA encoding the protein of interest to the hormone-binding domain of the estrogen receptor. In this case, tamoxifen can be used to activate the protein as recently demonstrated for c-Myc (9).
Another group has used topical application of anti-progestin to induce expression of TGF-1 in the epidermis (284). In this system, a fusion protein of a truncated progesterone receptor and the yeast GAL4 transcription factor was expressed under the control of the loricrin promoter. Thus by engineering a GAL4 binding domain, normally absent in mammalian cells, upstream of the target gene, transcription can be activated in a tissue-specific and temporally controlled manner. Finally, doxycycline-mediated gene expression (148) was recently used to inducibly express the erbB2 oncogene in the epidermis of transgenic mice (298).
Use of these types of systems will circumvent the problem of systemic defects and will also prevent abnormal skin development. The latter is important, since a wound healing phenotype might be secondary to a defect already present in nonwounded skin. Thus by induction of a transgene or deletion of an endogenous gene before wounding, the role of this particular gene in the healing response can be studied in the absence of secondary abnormalities. This type of study will undoubtedly unravel many exciting functions of growth factors and cytokines in normal wound repair as well as in impaired healing and scar formation.
We thank Dr. Gillian Ashcroft, Dr. Manfred Blessing, Dr. Jeffrey Davidson, Dr. Reinhard Gillitzer, Dr. Francesca Levi-Schaffer, and Dr. Paul Martin for many helpful suggestions and critical comments regarding the manuscript and Peter Gallasz for help with the figures.
Work in the laboratory of S. Werner is supported by the ETH Zürich, Swiss National Science Foundation Grant 31–61358.00, the Swiss and German Ministries for Education and Research, and the Stiftung Verum.
Address for reprint requests and other correspondence: S. Werner, Institute of Cell Biology, ETH Zurich, Hönggerberg, HPM D42, CH-8093 Zurich, Switzerland (E-mail: [email protected]).