EUROPEAN  TISSUE  REPAIR  SOCIETY

GROWTH FACTOR BIOLOGY AND TISSUE REPAIR
Sabine Werner and co-workers
Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland

Sabine Werner

Injury to adult tissues initiates a series of events including inflammation, new tissue formation and tissue remodelling. These processes are controlled by cell-cell and cell-matrix interactions as well as by a wide variety of soluble mediators. We are particularly interested in the role of growth factors and cytokines in the tissue repair process, and we study their molecular mechanisms of action. In addition, we have recently identified several novel genes which are regulated by cutaneous injury. The functional characterization of these genes with particular emphasis on their roles in tissue repair is another focus of our work.

The group was founded in 1993 at the Max-Planck-Institute of Biochemistry in Martinsried near Munich, Germany. In February 1999 we moved to the Institute of Cell Biology at the Swiss Federal Institute of Technology in Zürich, Switzerland. Our team currently includes seven postdoctoral scientists, seven Ph.D. students and four technical assistants. They use molecular methods, cell culture and animal models to answer the following questions:

• What is the role of keratinocyte growth factor in epithelial repair processes?
• What are the molecular mechanisms of keratinocyte growth factor action?
• What is the role of activin in tissue repair processes and inflammatory disease?
• What are the molecular mechanisms of activin action in keratinocytes?
• What is the role of cytoprotective molecules in wound repair?
• Which genes are regulated by skin injury and what are their roles in tissue repair?

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family of mitogens (FGF-7), is a secreted protein which is predominantly produced by various types of mesenchymal, but not by epithelial cells. However, most types of epithelial cells express the only known high-affinity receptor for KGF, a transmembrane protein tyrosine kinase.

We and others demonstrated a strikingly increased expression of KGF upon injury to adult skin. By contrast, the KGF receptor was exclusively expressed on kera-tinocytes of the epidermis and the hair follicles, suggesting 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 animals had a severe delay in wound reepithelia-lization, demonstrating the importance of KGF receptor signalling during cutaneous wound repair. Surprisingly, results from another laboratory demonstrated that mice lacking KGF have no obvious 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, since this new FGF family member is

(i) highly homologous to KGF,
(ii) co-expressed with KGF in adult mouse tissues (Beer et al, 1997), and
(iii) a high-affinity ligand of the KGF receptor.

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.

To gain insight into the mechanisms of KGF and FGF-10 action in normal and wounded skin we searched for genes which are regulated by these factors in cultured keratinocytes. We identified several unknown genes but also various known genes which 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 functional characterization of these genes is one of the major ongoing projects. We are studying their regulation, the biochemical properties of the encoded proteins, and particularly their roles in cutaneous wound healing and other types of repair processes.

Project 2: Activin: A novel player in inflammation and repair

Activins are members of the transforming growth factor-b superfamily of growth and differentiation factors. They influence proliferation and differentiation of many different cell types, but a role of activin in the skin has only recently been demonstrated.

To gain insight into the function of activin in normal and wounded skin, we analyzed the expression of activin and its receptors during cutaneous wound repair in mice. Activin expression was hardly detectable in non-wounded back skin. However, we found a strikingly increased expression of activin in the granulation tissue and in supra-basal keratinocytes of the hyperproliferative 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. These results suggested a dual function of activin in the granulation tissue and also in the differentiating keratinocytes.

To determine the activities of activin in the skin, we overexpressed the activin bA chain in the basal keratino-cytes of the epidermis of transgenic mice. These animals revealed significant abnormalities in the skin. The epidermis was much thicker compared to the skin of control animals due to hyperproliferation of the basal cells and abnormal keratinocyte differentiation

Apart from epidermal changes, the subcutaneous fatty tissue was replaced by connective tissue in the transgenic mice. The most interesting finding, however, was the striking enhancement of the wound healing process in the activin-overexpressing mice, whereby granulation tissue formation was particularly stimulated. 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.

Taken together, our results provide the first evidence for an important role of activin in wound repair as well as in keratinocyte differentiation, dermal fibrosis, and possibly also in human skin disease. We are currently analyzing the role of endogenous activin in the wound repair process and we are studying its molecular mechanisms of action in keratinocytes.

In addition to the skin, we have provided evidence for an important role of activin in other types of inflammatory and repair processes. Thus we have shown a strong overexpression of activin in affected areas of patients suffering from inflammatory bowel disease. Finally, in a collaboration with the laboratory of Dr Christian Alzheimer at the University of Munich we recently demonstrated a novel and important role of activin in repair processes of the central nervous system. We are currently studying the mechanisms of activin action in this process.