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Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, SwitzerlandMax-Planck-Institute of Biochemistry, Martinsried, Germany
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, SwitzerlandMax-Planck-Institute of Biochemistry, Martinsried, Germany
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, SwitzerlandMax-Planck-Institute of Biochemistry, Martinsried, Germany
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, SwitzerlandMax-Planck-Institute of Biochemistry, Martinsried, Germany
Reepithelialization and granulation tissue formation during cutaneous wound repair are mediated by a wide variety of growth and differentiation factors. Recent studies from our laboratory provided evidence for an important role of keratinocyte growth factor (KGF) in the repair of the injured epithelium and for a novel function of the transforming growth factor-β superfamily member activin in granulation tissue formation. KGF is weakly expressed in human skin, but is strongly upregulated in dermal fibroblasts after skin injury. Its binding to a transmembrane receptor on keratinocytes induces proliferation and migration of these cells. Furthermore, KGF has been shown to protect epithelial cells from the toxic effects of reactive oxygen species. We have identified a series of KGF-regulated genes that are likely to play a role in these processes. In addition to KGF, activin seems to be a novel player in wound healing. Activin expression is hardly detectable in nonwounded skin, but this factor is highly expressed in redifferentiating keratinocytes of the hyperproliferative wound epithelium as well as in cells of the granulation tissue. To gain insight into the role of activin in wound repair, we generated transgenic mice that overexpress activin in basal keratinocytes of the epidermis. These mice were characterized by a hyperthickened epidermis and by dermal fibrosis. Most importantly, overexpression of activin strongly enhanced the process of granulation tissue formation, demonstrating a novel and important role of activin in cutaneous wound repair.
Cutaneous injury initiates a series of events including inflammation, reepithelialization, and granulation tissue formation, and finally matrix remodeling. These processes are controlled by a multitude of cell–cell and cell–matrix interactions, but also by various growth and differentiation factors (reviewed by
). Although expression of many of these factors has been described in the healing wound, their precise roles and mechanisms of action have been poorly defined. Members of the fibroblast growth factor (FGF) and transforming growth factor (TGF)-β families have been shown to be key players in this process. Thus recent studies from our laboratory demonstrated an important role of keratinocyte growth factor (KGF, FGF-7) and its receptor in the repair of the injured epithelium, as well as a dual role of the TGF-β superfamily member activin in keratinocyte differentiation and particularly in granulation tissue formation. Here we report on the expression and function of these growth and differentiation factors in normal skin and particularly during cutaneous wound repair.
as a lung fibroblast-derived mitogen for mouse keratinocytes. It is a monomeric glycoprotein of 26–28 kDa that is produced by various types of mesenchymal cells in vitro and in vivo (reviewed by
). KGF expression has as yet not been detected in cells of epithelial origin; however, most types of epithelial cells express FGFR2-IIIb, the only known high-affinity receptor for KGF (
). These results suggested that KGF acts predominantly in a paracrine manner. Such a paracrine action of KGF seems to occur in normal and particularly in wounded skin. We and colleagues demonstrated a weak expression of KGF in murine and human skin; however, expression of this mitogen was strikingly induced in dermal fibroblasts upon skin injury (
). By contrast, the KGF receptor was exclusively expressed on keratinocytes of the epidermis and the hair follicles. This expression pattern of KGF and its receptor suggested that dermally derived KGF stimulates wound reepithelialization in a paracrine manner. This hypothesis was strongly supported by the wound healing phenotype seen in transgenic mice, which express a dominant-negative KGF receptor in the basal keratinocytes of the epidermis and in the outer root sheath keratinocytes of the hair follicles. These mice were characterized by a severe delay in wound reepithelialization (
), demonstrating the importance of KGF receptor signaling during cutaneous wound repair. Surprisingly, mice lacking KGF revealed no phenotypic abnormalities and even the healing process of incisional wounds appeared normal (
), suggesting that other KGF receptor ligands can compensate for the lack of KGF. The most likely candidate for such a compensatory effect is FGF-10, which is highly homologous to KGF (
). As shown in Figure 1, a very similar expression pattern of KGF (FGF-7) and FGF-10 was observed in adult mouse tissues, whereby both factors were expressed at particularly high levels in the lung and the skin. Interestingly, they are coexpressed with their only high-affinity receptor (FGFR2-IIIb) in these tissues (Figure 1, lower panel), indicating that their strong expression is functionally important. These data suggest that FGF-10 can indeed compensate for the lack of FGF-7 in a knockout mouse. We also found expression of FGF-10 in wounded skin, although it was not upregulated at the transcriptional level; however, the levels of bioactive FGF-10 in the skin might be increased by wounding, as FGF-10 was shown to be cell associated in vitro and thus might be released from the cells upon injury (
). In summary, these results demonstrated an important role of KGF receptor ligands in wound repair, although the precise function of each ligand in this process remains to be determined.
Figure 1Expression of KGF (FGF-7), FGF-10, and FGFR2-IIIb (KGF receptor) in adult mouse tissues. Total cellular RNA was isolated from adult mouse tissues; 20 μg RNA was analyzed for the expression of KGF (FGF-7), FGF-10, and FGFR2-IIIb by RNase protection assay; 20 μg tRNA were used as a negative control; 1000 cpm of the hybridization probes were loaded in the lanes labeled "probe" and used as a size marker. The same set of RNA was used for all protection assays.
and from Beer et al. (J Biol Chem 275:16091–16097 2000) with permission from Stockton Press and from the American Society for Biochemistry of Molecular Biology.
To gain insight into the mechanisms of KGF and FGF-10 action in normal and wounded skin we searched for genes that are regulated by these factors in cultured keratinocytes. For this purpose we used the differential display reverse transcriptase-polymerase chain reaction technology and we also generated a subtractive cDNA library from quiescent and KGF-treated keratinocytes. Using these technologies we identified a wide variety of KGF-regulated genes. Besides several unknown genes we also identified various known genes that are summarized in Figure 2. These genes might help to explain how KGF mediates various processes in normal and wounded skin, such as keratinocyte proliferation and migration, protection from the toxic effects of reactive oxygen species, and indirect effects such as angiogenesis and stimulation of matrix deposition. The KGF-regulated genes and their possible functions in normal and wounded skin are described below.
Figure 2Overview of KGF-regulated genes. The products of the genes shown on the right-hand side are likely to mediate the direct effects of KGF, whereas those on the left-hand side could be responsible for the indirect effects of KGF on mesenchymal cells.
Genes that are involved in the regulation of keratinocyte proliferation and migration
Various genes were identified that are likely to be involved in the mitogenic effect of KGF. One of the genes encodes adenylosuccinate lyase, an enzyme involved in purine de novo synthesis and in the salvage pathway (
). To determine whether regulation of enzymes involved in nucleotide biosynthesis is a general mechanism of KGF action, we cloned cDNA probes of the key regulatory enzymes involved in this metabolic pathway, including adenylosuccinate synthetase, the enzyme responsible for converting inosine monophosphate into adenylosuccinate; phosphoribosyl pyrophosphate synthetase, which generates phosphoribosyl pyrophosphate, a molecule required for de novo production of both purine and pyrimidine nucleotides and for the purine salvage pathway; amidophosphoribosyl transferase, a key regulatory enzyme in purine biosynthesis; hypoxanthine (guanine) phosphoribosyl transferase (HPRT), an important enzyme of the salvage pathway, and of the multifunctional protein CAD (carbamoylphosphate synthetase II, aspartate transcarbamylase, dihydroorotase). Interestingly, the expression of all of these enzymes was strongly induced by KGF in cultured keratinocytes, as shown in Figure 3(a) for HPRT. This regulation is likely to be important for the in vivo situation, because we found high levels of the corresponding transcripts in healing skin wounds (Figure 3b), particularly in the KGF-responsive keratinocytes of the hyperproliferative epithelium at the wound edge (
; Figure 3b). These findings suggest that KGF also stimulates the synthesis of enzymes involved in nucleotide biosynthesis during wound healing, which should allow the production of sufficient amounts of nucleotides required for DNA replication and RNA synthesis.
Figure 3Increased expression of HPRT by KGF in vitro and by cutaneous injury in vivo. (A) KGF-regulated expression of HPRT in HaCaT keratinocytes. Quiescent HaCaT keratinoytes were incubated with KGF for different time periods as indicated; 20 μg total cellular RNA were analyzed for the expression of HPRT; 20 μg tRNA were used as a negative control; 1000 cpm of the different hybridization probe was loaded in the lane labeled ‘‘probe’' and used as a size marker. (B) Expression of HPRT during cutaneous wound repair in mice. Full-thickness excisional wounds were generated on the back of BALB/C mice. Mice were sacrificed at different time points after injury. The complete wounds were isolated and used for RNA isolation; 20 μg total cellular RNA from normal and wounded skin were analyzed by RNase protection assay for the expression of HPRT; 20 μg tRNA were used as a negative control; 1000 cpm of the hybridization probe was loaded in the lane labeled ‘‘probe’' and used as a size marker. (C) Expression of HPRT in 5 day full-thickness excisional wounds. Frozen sections from the middle of full-thickness excisional wounds were hybridized with a 35S-labeled antisense riboprobe complementary to the murine HPRT. Scale bar: 1 μm. Signals appear as white dots in the dark field survey. ES, Eschar; HE, hyperproliferative epithelium; G, granulation tissue. The figure has been adopted from
In addition to these nucleotide biosynthesis enzymes, we recently identified a putative human homolog of the yeast CHL-1 gene as a KGF-regulated gene (
), suggesting that a human homolog might have a similar function in keratinocytes. Finally, we found upregulation of the transcription factor c-myc in response to KGF and downregulation of a putative novel transcription factor (KRG-3, KGF-regulated gene 3) (H.-D.B., unpublished data). Preliminary results from our laboratory suggest a role of the latter in the regulation of keratinocyte differentiation, indicating that downregulation of a differentiation-inducing factor might play a role in the mitogenic effect of KGF.
With regard to the stimulatory effect of KGF on keratinocyte migration, we found a KGF-mediated increase in the expression of the matrix metalloproteinase stromelysin-2 (
). The latter is normally expressed in migrating keratinocytes at the wound edge where it is likely to be responsible for the degradation of the provisional matrix, a function that is essential for migration of keratinocytes into the wound (reviewed by
An important feature of KGF is its protective effect for various types of epithelial cells. Thus, recombinant KGF was successfully used as a pretreatment in several mouse models of gastrointestinal injury induced by radiation, chemotherapy, or a combination of both. In the bladder, injection of KGF before cyclophosphamide prevented the ulcerative hemorrhagic cystitis that is normally induced by this drug, and in the lung, exogenous KGF prevented lung injury in various model systems (reviewed by
). Finally, recent results from our laboratory suggest that KGF can reduce oxidative damage in keratinocytes. These observations suggested that KGF can induce the expression of enzymes involved in the detoxification of reactive oxygen species. To test this possibility, we analyzed the effect of KGF on the expression of phospholipid hydroperoxide glutathione peroxidase, as well as catalase and the Mn-and Cu/Zn-dependent superoxide dismutases. As shown in Figure 4, none of these enzymes was regulated by KGF at the transcriptional level. We identified a nonselenium glutathione peroxidase, however, as a novel KGF-regulated gene using the differential display reverse transcriptase-polymerase chain reaction technology (
). Expression of this enzyme was only induced by KGF but not by other growth factors in keratinocytes, suggesting that this effect is specific for KGF. The upregulation of this enzyme by KGF is likely to be important in vivo, because the enzyme was strongly overexpressed in the hyperproliferative epithelium of murine skin wounds (
). These results suggested that KGF reduces the toxic effects of reactive oxygen species at least in part by upregulation of this enzyme. In addition, we recently demonstrated increased expression of DNA repair enzymes by KGF in vitro, demonstrating that KGF regulates various genes that protect epithelial cells from environmental damage.
Figure 4Effect of KGF on the expression of enzymes involved in the detoxification of reactive oxygen species. Quiescent HaCaT keratinocytes were treated with KGF for different time periods as indicated; 20 μg RNA isolated from these cells were analyzed by RNase protection assay for the expression of the nonselenium glutathione peroxidase (nsGPx), the phospholipid hydroperoxide glutathione peroxidase (PhGPx), catalase (CAT), the Mn-dependent superoxide dismutase (Mn-SOD), and the Cu/Zn-dependent superoxide dismutase (Cu/Zn-SOD); 20 μg tRNA were used as a negative control; 1000 cpm of the hybridization probes were loaded in the lanes labeled ‘‘probe’' and used as a size marker. The same set of RNA was used for all protection assays. The RNase protection assay showing nsGPx expression was adopted from
demonstrated indirect effects of exogenously applied KGF or FGF-10, respectively, on the granulation tissue. Thereby, increased collagen deposition was observed in both studies. Because KGF is likely to act only on epithelial cells in the wound, the observed stimulation of granulation tissue formation might be a result of other factors that are upregulated by KGF in keratinocytes. Indeed, we previously demonstrated a striking upregulation of vascular endothelial growth factor expression by KGF in cultured HaCaT keratinocytes (
), suggesting that KGF can indirectly stimulate angiogenesis via VEGF induction. Furthermore, we recently demonstrated induction of TGF-β1 and activin expression by KGF in cultured keratinocytes (
; Frank and Werner, unpublished data). Because both of these factors stimulate production of extracellular matrix molecules in fibroblasts in vitro and in vivo (reviewed by
Overexpression of activin in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.
; see below), they might be at least partially responsible for the increased collagen deposition seen in KGF-treated wounds. The potent stimulation of activin expression by KGF in keratinocytes tempted us to speculate about a novel role of activin in wound repair. Indeed, this hypothesis has recently been confirmed, and the results that we have obtained with activin will be summarized below.
Activin
Activins, which belong to the TGF-β superfamily of growth and differentiation factors, are dimeric proteins, the monomeric polypeptides of which are connected by disulfide linkage. Three different forms of activin, activin A (βAβA), activin B (βBβB), and activin AB (βAβB), have been characterized (reviewed by
), although the function of the corresponding proteins is as yet unknown. Activins influence proliferation and differentiation of many different cell types (reviewed by
), but a role of activin in normal and wounded skin has only recently been demonstrated. The first evidence for a possible role of activin in the skin resulted from the phenotypes of knockout mice. Besides various defects that caused death within the first 24 h after birth, activin βA knockout mice lacked whiskers and whisker follicles were abnormal (
To gain insight into the function of activin in normal and wounded skin, we recently analyzed the expression of activin and its receptors during cutaneous wound repair in mice. Activin expression was hardly detectable in nonwounded back skin; however, we found a strikingly increased expression of activin βA and βB mRNA in the granulation tissue and in suprabasal keratinocytes of the hyper- proliferative epithelium after skin injury (
). Furthermore, all known activin receptors were expressed in the mesenchymal and epithelial compartments of normal and wounded skin, although their expression did not change after injury (
Overexpression of activin in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.
To determine the activities of activin in the skin, we overexpressed the activin βA chain in the epidermis of transgenic mice under the control of a keratin 14 promoter that targets the expression of transgenes to the nondifferentiated basal cells of the epidermis and to the keratinocytes of the outer root sheath of the hair follicles (
; Ongena and Huylebroeck, unpublished data). The transgenic mice revealed significant abnormalities in the skin. The epidermis was much thicker compared with the skin of control animals (Figure 5b). The spinous layer was greatly increased compared with normal epidermis, but little parakeratosis was observed in the cornified layer. Furthermore, the epidermal architecture was highly disorganized. Consistent with the appearance of the epidermis, the rate of proliferating basal keratinocytes was 2.5-fold higher in the skin of transgenic animals compared with the controls. This stimulation of keratinocyte proliferation by activin is likely to be indirect, as activin inhibited proliferation of human HaCaT keratinocytes (
Figure 5Expression of the basal keratin 14 in the tail skin of normal and activin-overexpressing mice. Six micrometer frozen sections were stained with a monospecific antiserum directed against keratin 14 and a rhodamine-coupled secondary antibody. Note the severe hyperthickening of the epidermis of the transgenic mice (A) and the strong expression of keratin 14 in basal cells of the epidermis as well as in outer root sheath keratinocytes of the hair follicles of normal (B) and activin-overexpressing mice (A).
We subsequently determined the stage of differentiation of the additional nucleated suprabasal cells. Interestingly, the basal cell keratin 14 (Figure 5) and the differentiation-specific keratin 10 were expressed in the appropriate layers in normal and transgenic mice, demonstrating that the additional layers seen in the epidermis of the transgenic animals consist of at least partially differentiated cells. These cells did not express markers of terminal differentiation, however, indicating that activin allows or even stimulates the early differentiation process, whereas terminal differentiation seems to be delayed. A role of activin in the early induction of keratinocyte differentiation is further supported by the expression of activin in the suprabasal but not in the basal cells of the hyperproliferative wound epithelium (
and unpublished data). Induction of keratinocyte differentiation by activin A has also been shown in primary keratinocytes in vitro (Seishima et al. 1999). In contrast to our in vivo results, however, the process of terminal differentiation was also enhanced by activin in these cells. These discrepancies could be due to the presence of other factors present in vivo in a hyperproliferative epithelium that delay the later differentiation steps.
Apart from epidermal changes, the subcutaneous fatty tissue was replaced by connective tissue in the transgenic mice. Therefore, we analyzed the expression of various extracellular matrix proteins in these animals. Although no significant differences in the mRNA expression levels of fibronectin, collagen βI, collagen βIII, and of the basement membrane proteins tenascin-C and nidogen were observed, we found a strong deposition of tenascin-C in the interfollicular dermis of the transgenic animals, whereas this protein is normally restricted to the basement membrane zones. These results suggest that activin gradually alters extracellular matrix molecule metabolism, probably by diffusion from the keratinocytes into the underlying dermis.
Finally, we generated full-thickness excisional wounds on the back of transgenic mice and control litter mates, and we performed a detailed histologic analysis of 5 day wounds from three independent transgenic mouse lines that express high levels of the transgene. Interestingly, the wound healing process in the activin-overexpressing mice was strongly enhanced, whereby granulation tissue formation was particularly stimulated. Large amounts of extracellular matrix were detected below the activin-producing keratinocytes, indicating that activin stimulates connective tissue synthesis/deposition not only in normal but also in wounded skin. These data suggest a novel role of activin in fibrotic processes and this hypothesis is strongly supported by the detection of high activin levels in various types of fibrotic disease (reviewed by
Taken together, our results provide first evidence for an important role of activin in wound repair as well as in keratinocyte differentiation, in dermal fibrosis, and possibly also in human skin disease.
ACKNOWLEDGMENTS
We thank Andreas Stanzel and Céline Mamie for excellent technical assistance. Work in our laboratory is supported by a Human Frontier Science Grant, the German Ministry for Education and Research, and the Stiftung Verum.
References
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Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression.
Overexpression of activin in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.
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