Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double-knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor

Mechanistic assessment of fibroblast growth factor receptor signaling and function in keratinocytes

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

The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis

Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae

Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice

FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with re-markable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 acti-vation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of re-sponsiveness to light activation. These results demonstrate un-expected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.ISSN:2575-107

Antagonism of interferon signaling by fibroblast growth factors promotes viral replication

Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross-talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon-stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF-mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV-1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV-1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections

FGFR3 overactivation in the brain is responsible for memory impairments in Crouzon syndrome mouse model

International audienceCrouzon syndrome with acanthosis nigricans (CAN, a rare type of craniosynostosis characterized by premature suture fusion and neurological impairments) has been linked to a gain-of-function mutation (p.Ala391Glu) in fibroblast growth factor receptor 3 (FGFR3). To characterize the CAN mutation's impact on the skull and on brain functions, we developed the first mouse model (Fgfr3 A385E/+) of this syndrome. Surprisingly, Fgfr3 A385E/+ mice did not exhibit craniosynostosis but did show severe memory impairments, a structurally abnormal hippocampus, low activity-dependent synaptic plasticity, and overactivation of MAPK/ERK and Akt signaling pathways in the hippocampus. Systemic or brain-specific pharmacological inhibition of FGFR3 overactivation by BGJ398 injections rescued the memory impairments observed in Fgfr3 A385E/+ mice. The present study is the first to have demonstrated cognitive impairments associated with brain FGFR3 overactivation, independently of skull abnormalities. Our results provide a better understanding of FGFR3's functional role and the impact of its gain-of-function mutation on brain functions. The modulation of FGFR3 signaling might be of value for treating the neurological disorders associated with craniosynostosis