Richard Grose

Fibroblast growth factor signalling: from development to cancer

Fibroblast growth factors (FGFs) and their receptors control a wide range of biological functions, regulating cellular proliferation, survival, migration and differentiation. Although targeting FGF signalling as a cancer therapeutic target has lagged behind that of other receptor tyrosine kinases, there is now substantial evidence for the importance of FGF signalling in the pathogenesis of diverse tumour types, and clinical reagents that specifically target the FGFs or FGF receptors are being developed. Although FGF signalling can drive tumorigenesis, in different contexts FGF signalling can mediate tumour protective functions; the identification of the mechanisms that underlie these differential effects will be important to understand how FGF signalling can be most appropriately therapeutically targeted.

Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes

β1 integrins are ubiquitously expressed receptors that mediate cell–cell and cell–extracellular matrix interactions. To analyze the function of β1 integrin in skin we generated mice with a keratinocyte‐restricted deletion of the β1 integrin gene using the cre–loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of α6β4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin‐5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal–epidermal junction. In contrast, the integrity of the basement membrane surrounding the β1‐deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of β1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes.

The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing

Endostatin is a cleavage product of collagen XVIII that strongly inhibits tumor angiogenesis. To determine if endostatin affects other angiogenic processes, we generated full‐thickness excisional wounds on the back of mice that were systemically treated with recombinant murine endostatin. No macroscopic abnormalities of the wound healing process were observed. Histological analysis revealed normal wound contraction and re‐epithelialization, but a slight reduction in granulation tissue formation and reduced matrix deposition at the wound edge. The blood vessel density in the wounds of endostatin‐treated mice was not affected. However, ultrastructural analysis demonstrated severe abnormalities in blood vessel maturation. The wound vessels in the endostatin‐treated mice were narrowed or closed with an irregular luminal surface, resulting in a severe reduction in the number of functional vessels and extravasation of erythrocytes. Endostatin treatment did not affect the expression level and localization of collagen XVIII mRNA and protein. Furthermore, the angiogenesis regulators vascular endothelial growth factor, angiopoietin‐1, and angiopoietin‐2 were normally expressed in the wounds of endostatin‐treated mice. However, expression of the major wound matrix proteins fibronectin and collagens I and III was significantly reduced. This reduction is likely to explain the reduced density of the wound matrix. Our results demonstrate that endostatin treatment reduces the number of functional blood vessels and the matrix density in the granulation tissue, but does not significantly affect the overall wound healing process.

Wound-healing studies in transgenic and knockout mice.

Injury to the skin initiates a cascade of events including inflammation, new tissue formation, and tissue remodeling, that finally lead to at least partial reconstruction of the original tissue. Historically, animal models of repair have taught us much about how this repair process is orchestrated and, over recent years, the use of genetically modified mice has helped define the roles of many key molecules. Aside from conventional knockout technology, many ingenious approaches have been adopted, allowing researchers to circumvent such problems as embryonic lethality, or to affect gene function in a tissue-or temporal-specific manner. Together, these studies provide us with a growing source of information describing, to date, the in vivo function of nearly 100 proteins in the context of wound repair.This article focuses on the studies in which genetically modified mouse models have helped elucidate the roles that many soluble mediators play during wound repair, encompassing the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) families and also data on cytokines and chemokines. Finally, we include a table summarizing all of the currently published data in this rapidly growing field. For a regularly updated web archive of studies, we have constructed a Compendium of Published Wound Healing Studies on Genetically Modified Mice which is available at http://icbxs.ethz.ch/members/grose/woundtransgenic/home.html.

A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning

To understand the role Fgf signalling in skin and hair follicle development, we analysed the phenotype of mice deficient for Fgfr2-IIIb and its main ligand Fgf10. These studies showed that the severe epidermal hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast, Fgfr2-IIIb–/–, but not Fgf10–/–, mice produced significantly fewer hair follicles, and their follicles were developmentally retarded. Following transplantation onto nude mice, grafts of Fgfr2-IIIb–/– skin showed impaired hair formation, with a decrease in hair density and the production of abnormal pelage hairs. Expression of Lef1, Shh and Bmp4 in the developing hair follicles of Fgfr2-IIIb–/– mice was similar to wild type. These results suggest that Fgf signalling positively regulates the number of keratinocytes needed to form a normal stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs.

The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells.

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling.

The upregulation of TGF-β1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking β3-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-β1 and enhanced dermal fibroblast infiltration into wounds of β3-null mice. Specifically, β3-integrin deficiency is associated with elevated TGF-β receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-β1-mediated dermal fibroblast migration. These data indicate that αvβ3–integrin can suppress TGF-β1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-β1 regulation by β3-integrins.

E‐cadherin controls adherens junctions in the epidermis and the renewal of hair follicles

E‐cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E‐cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E‐cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and β‐catenin levels were not strongly altered in the mutant skin. We conclude that E‐cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E‐cadherin.

Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis

Loss of FGFRs results in skin abnormalities due to activation of keratinocytes and epidermal T cells.

The role of fibroblast growth factor receptor 2b in skin homeostasis and cancer development

The epithelial isoform of fibroblast growth factor receptor 2 (Fgfr2b) is essential for embryogenesis, and Fgfr2b‐null mice die at birth. Using Cre‐Lox transgenics to delete Fgfr2b in cells expressing keratin 5, we show that mice lacking epidermal Fgfr2b survive into adulthood but display striking abnormalities in hair and sebaceous gland development. Epidermal hyperthickening develops with age, and 10% of mutant mice develop spontaneous papillomas, demonstrating the role of Fgfr2b in post‐natal skin development and in adult skin homeostasis. Mice lacking epithelial Fgfr2b show great sensitivity to chemical carcinogenic insult, displaying several oncogenic ha‐ras mutations with dramatic development of papillomas and squamous cell carcinomas. Mutant mice have increased inflammation in the skin, with increased numbers of macrophages and γδT cells with abnormal morphology. Mutant skin shows several changes in gene expression, including enhanced expression of the pro‐inflammatory cytokine interleukin 18 and decreased expression of Serpin a3b, a potential tumor suppressor. Thus we describe a novel role of Fgfr2b and provide the first evidence of a tyrosine kinase receptor playing a tumor suppressive role in the skin.