EUROPEAN TISSUE REPAIR SOCIETY

GROWTH FACTORS I

KERATINOCYTE GROWTH FACTORS: IMPORTANT PLAYERS IN EPITHELIAL REPAIR PROCESSES

CUTANEOUS injury initiates a series of events including inflammation, granulation tissue formation, reepithelialisation, and finally matrix remodelling. These processes are controlled by cell-cell and cell-matrix interactions and also by various growth and differentiation factors.¹ Expression of many of these factors has been described in the healing skin wound, but their roles in the repair process are poorly defined. Members of the fibroblast growth factor (FGF) family have been shown to be key regulators of wound healing. In particular, our laboratory demonstrated an important role of keratinocyte growth factor (KGF, FGF-7) and its receptor in the repair of the injured epidermis.

KGF was discovered as a lung fibroblast-derived mitogen for murine keratinocytes. It is a secreted glycoprotein which is produced by various types of mesenchymal cells in vitro and in vivo as well as by γδ T-cells in inflamed tissues.² KGF expression has not as yet been detected in epithelial cells. However, most types of epithelial origin express FGFR2-IIIb (fibroblast growth factor receptor), the only known high-affinity receptor for KGF,³ and these cells have been shown to be activated by KGF.² This indicated 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 others have demonstrated a weak expression of KGF in murine and human skin. However, expression of this growth factor was strongly induced in dermal fibroblasts upon skin injury^4,5 (Figure 1A and B). By contrast, FGFR2-IIIb was exclusively expressed on keratinocytes of the epidermis and the hair follicles (Figure 1B). This expression pattern of KGF and its receptor suggested that dermally derived KGF stimulates wound reepithelialisation in a paracrine manner (Figure 2). This hypothesis was strongly supported by the phenotype observed in transgenic mice which express a dominant-negative KGF receptor mutant in the basal keratinocytes of the epidermis and in outer root sheath keratinocytes of hair follicles. These animals were characterized by a severe reduction in keratinocyte proliferation in normal and particularly in wounded skin, resulting in a major delay in wound reepithelialisation6 (Figure 1C). Thus, KGF receptor signalling was shown to play an important role in wound repair. This result is supported by the stimulatory effect of exogenous KGF on the wound healing process in various animal models.² Surprisingly, full-thickness incisional skin wounds healed normally in KGF knockout mice,⁷ 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 and which binds to the KGF receptor with high affinity^8,9 (Figure 2). A very similar expression pattern of KGF and FGF-10 was observed in adult mouse tissues, with both factors being expressed at particularly high levels in mesenchymal cells of the lung and the skin. Interestingly, co-expressed in the epithelial cells of these tissues is their only high-affinity receptor (FGFR2-IIIb), indicating that their strong expression is functionally important.10 These data suggest that FGF-10 may indeed compensate for the lack of KGF in a KGF-null 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 are likely to be elevated in injured skin, since FGF10 is cell-associated in vitro and thus might be released from the cell’s surface after wounding.10 Although FGF-10 only binds to the epithelial-specific FGFR2-IIIb with high affinity, it was also shown to bind to another type of FGF receptor, FGFR1-IIIb, with lower affinity (Figure 2). This type of receptor was identified and cloned in our laboratory^9,11 and found to be expressed at particularly high levels in the skin.11 In contrast to FGFR2-IIIb, FGFR1-IIIb is not only expressed by epithelial cells but also by fibroblasts.¹¹ This finding might explain why exogenous FGF-10 not only stimulates reepithelialisation, but also granulation tissue formation.¹² Thus, FGF-10, which is currently in clinical trials for the treatment of venous ulcers,¹³ is likely to enhance reepithelialisation via the activation of FGFR2-IIIb on keratinocytes and granulation tissue formation via it’s binding to FGFR1-IIIb on fibroblasts. Finally, a third member of the FGF family, FGF- 22, with high homology to KGF and FGF-10, has recently been discovered and found to be expressed by hair follicle keratinocytes.¹⁴ Studies from our laboratory revealed that expression of FGF-22 is upregulated in wounded mouse skin. In contrast to KGF and FGF-10, FGF-22 is expressed by keratinocytes, suggesting that it acts in an autocrine manner to stimulate wound reepithelialisation (R.G. and S.W., unpublished data).

Figure 1. Expression and function of KGF and its receptor in wound healing.

A: Increased expression of KGF in human incisional wounds. Total cellular RNA was isolated from normal skin and from incisional wounds of healthy adult volunteers at different time points after injury. 10 mg RNA was analyzed for the expression of KGF by RNase protection assay. 20 mg tRNA were used as a negative control. 1000 cpm of the hybridisation probes were loaded in the lane labeled ‘probe’ and used as a size marker.

B: Schematic representation of KGF and KGF receptor expression in a skin wound. KGF is produced by fibroblasts in the dermis and the granulation tissue (blue) and acts in a paracrine manner to stimulate proliferation and migration of keratinocytes which express FGFR2-IIIb, the high affinity KGF receptor (pink).

C: Expression of a dominant-negative FGFR2-IIIb mutant in the epidermis of transgenic mice inhibits wound reepithelialisation. Paraffin sections from the middle of 5-day wounds of control mice (wt, on the left) and mice expressing a dominant-negative FGFR2-IIIb in the epidermis (dnKGFR, on the right) were stained with hematoxylin/ eosin. Note the reduced thickness of the wound epidermis in the transgenic animals. e: wound epidermis, Es: eschar, g: granulation tissue, h: hair follicle. Fig. 1C was reprinted with permission from ‘S. Werner et al. The function of KGF in morphogenesis of epithelium and reepithelialisation of wounds. Science 266, 819-822, 1994’. ©1994 American Association for the Advancement of Science.

Figure 2. Expression pattern and receptor binding of KGF, FGF-10 and FGF-22.

FGFR2-IIIb is expressed on epithelial cells and binds KGF (FGF-7) and FGF-10 with high affinity. FGFR1- IIIb is expressed on epithelial cells, fibroblasts and other cell types such as neurons and binds FGF-10 with low affinity. Due to its sequence homology with KGF and FGF-10, FGF-22 might also activate FGFR2-IIIb and/or FGFR1-IIIb.

Taken together, studies from our group and from others suggest that KGF and its homologues FGF-10 and FGF- 22 are involved in epithelial repair during skin wound healing. Most interestingly, these growth factors are likely to play important roles in the repair of other epithelial tissues. Thus, induction of KGF expression occurs after injury to the lung, the kidney, the bladder and the intestine.2 Recently, upregulation of KGF expression in a dextransulfate sodium (DSS)-induced colitis model in mice was found to be functionally important, since DSS-induced intestinal injury was more pronounced in KGF knockout mice, and epithelial repair upon ablation of KGF was delayed in these animals.¹⁵ Finally, recent results from our laboratory indicate that KGF-receptor signalling is crucial for liver regeneration after partial hepatectomy (H.S. and S.W., unpublished data). Thus, the KGF receptor and its ligands appear to be major regulators of epithelial repair in various tissues and organs.

References

1. Martin P. Wound healing: aiming for perfect skin regeneration. 1997; Science 276, 75–81.
2. Werner S. Keratinocyte growth factor: A unique player in epithelial repair processes. Cytokine Growth Factor Rev 1998; 9: 153–165.
3. Miki T, Fleming TP, Bottaro DP, Rubin JS, Ron D et al. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. 1991; Science 251: 72–75.
4. Werner S, Peters KG, Longaker MT, Fuller-Pace F, Banda M et al. Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc Natl Acad Sci USA 1992a; 89: 6896–6900.
5. Marchese C, Chedid M, Dirsch OR, Csaky KG, Santanelli et al. Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin. J Exp Med 1995; 182: 1369–1376.
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7. Guo L, Degenstein L and Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing. Genes and Dev 1996; 10: 165–175.
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10. Beer HD, Florence C, Dammeier J, McGuire L, Werner S et al. Mouse fibroblast growth factor 10: cDNA cloning, protein characterisation, and regulation of mRNA expression. Oncogene 1997; 15: 2211–18.
11. Werner S, Duan DS, de Vries C, Peters KG, Johnson D et al. Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligandbinding specificities. Mol Cell Biol 1992b; 12: 82–8.
12. Jimenez P and Rampy MA. Keratinocyte growth factor-2 accelerates wound healing in incisional wounds. J. Surg. Res. 1999; 81: 238–242.
13. Robson MC, Phillips TJ, Falanga V, Odenheimer DJ, Parish L et al. Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen. 2001; 9: 347–352.
14. Nakatake Y, Hoshikawa M, Asaki T, Kassai Y and Itoh N. Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle. Biochim. Biophys. Acta 2001; 1517: 460–463.
15. Chen Y, Chou K, Fuchs E, Havran WL and Boismenu R. Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc. Natl. Acad. Sci. USA 2002; 29: 14338–343.

Heike Steiling, Hans-Dietmar Beer, Richard Grose*,
Tobias A. Beyer, and Sabine Werner
Institute of Cell Biology, Department of Biology,
ETH Zürich, Hönggerberg,
CH–8093 Zürich, Switzerland
*Cancer Research UK, London Research Institute,
London WC2A 3PX, UK

Address for correspondence:
Dr Sabine Werner
Institute of Cell Biology
ETH Zürich, Hönggerberg
CH–8093 Zürich, Switzerland
Tel: +41 1 633 3941
Fax: +41 1 633 1174
E-mail: Sabine.werner@cell.biol.ethz.ch