Mechanistic assessment of fibroblast growth factor receptor signaling and function in keratinocytes
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2021
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Doctoral Thesis
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yes
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Abstract
The skin protects the body from environmental insults by forming an indispensable barrier. When it gets disrupted by wounding or upon development of certain skin diseases, different cell types have to act together to restore the barrier. Therefore, cellular communication via secretion of growth factors and other signaling molecules, which bind to receptors on the receiving cell, is essential. Major players in the communication between keratinocytes and fibroblasts, the main cell types of epidermis and dermis, respectively, are fibroblast growth factors (FGFs) and their receptors (FGFRs). However, FGFR signaling is complex and involves a large variety of ligands, which activate different splice variants of four high-affinity FGFRs. Additionally, the intracellular signal transduction molecules are not exclusive for FGF signaling, but are shared with and influenced by other signaling pathways. This complexity is a major obstacle when investigating effects of different FGFs and FGF receptors.
To reduce the complexity and to investigate the effect of only one FGFR, we modified the major FGFR expressed in keratinocytes, FGFR2, to be activated by blue light (OptoR2) instead of its endogenous ligands. In HEK 293T cells expressing OptoR2, illumination activated the downstream signaling pathways with remarkable temporal precision, and induced cell migration and proliferation. Keratinocytes expressing OptoR2 also responded to blue light with activation of their downstream pathways, regulation of known FGF target genes and increased migration. However, in all keratinocyte in vitro and in vivo systems that we generated, the cells lost the responsiveness to OptoR2 over time through receptor down-regulation or desensitisation to FGFR signaling. Therefore, a novel optogenetic strategy is required to further study FGFR signaling in the skin.
Another means to reduce complexity is removal of a single signaling component and analysis of the effect that this loss exerts in the cells. However, receptors and downstream signaling molecules are frequently redundant. Keratinocytes, for example, express FGFR1, FGFR2 and FGFR3. When FGFR1 and FGFR2 are lost in keratinocytes, mice develop a skin phenotype resembling Atopic Dermatitis in humans, with epidermal thickening, barrier defects and inflammation. These abnormalities are absent or reduced in mice lacking only a single FGFR. However, FGFR3 might compensate at least in part for the loss of the other two FGFRs. Therefore, we generated mice lacking FGFR1, FGFR2 and FGFR3 in keratinocytes to determine the effect of complete loss of FGFR signaling in this cell type. I contributed to the analysis of the phenotype of these mice, which revealed that loss of FGFR3 alone does not affect skin homeostasis and repair. Surprisingly, the phenotype of mice lacking all three receptors was not much stronger compared to the abnormalities seen in the absence of FGFR1 and FGFR2. It is possible that other receptor tyrosine kinases compensate for the loss of FGFR signaling in keratinocytes.
Finally, we discovered an unexpected interaction between FGF and type I interferon signaling, which depends on FGFR kinase and proteasomal activity, but not on type I interferon receptors. Consequently, FGF7-stimulated keratinocytes produce less antiviral proteins and are more prone to viral infection. Vice versa, inhibition of FGFR signaling strongly reduces the infection of keratinocytes with different viruses. This finding opens the possibility of treating viral infections with FGFR inhibitors, which should be further explored in the future.
Overall, my work contributed to the understanding of FGFR signaling at multiple levels and identified strengths and weaknesses of optogenetic approaches to study growth factor function. Finally, it opened potential new avenues for the treatment of viral infections in humans using FGFR kinase inhibitors.
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Examiner : Werner, Sabine
Examiner : Hynes, Nancy E.
Examiner : Detmar, Michael
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ETH Zurich
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03520 - Werner, Sabine / Werner, Sabine