Generation and analysis of conditional and inducible transgenic mouse models for analysis of cFLIP long and short isoforms in skin

• Applicant: Diana Panayotova Dimitrova, Ph.D.
• Subject Area: Dermatology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397983964

• Final Report Year: 2023

Final Report Abstract

In this follow-up project we have further investigated the role of the key molecule from the cell death signaling - cFLIP in the regulation of the cellular homeostasis and cell death in keratinocytes. We have generated several new epidermal mouse models namely, double KO cFLIP KO/ TNF KO, epidermal cFLIP long and cFLIP short expressing animals. We have performed both, in vivo analysis for the characterization of the modulated skin and in vitro studies with keratinocytes isolated from the experimental animals. Our data demonstrated that TNF signaling is critical factor for the regulation of skin inflammation in the absence of cFLIP. We could demonstrate that modulated cytokine and chemokine expression upon deletion of cFLIP is responsible for the inflammatory phenotype. Of interest, we have shown that that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells but is critical for neutrophil attraction. However, since the loss of TNF could not completely rescue, but only reduced and delayed the characteristic phenotype of cFLIP deficient skin, additional signaling pathways and control mechanisms are possibly involved. Taken together, we have confirmed that the inflammatory skin disease that results from the epidermal deletion of cFLIP is strongly but not exclusively dependent on TNF. Furthermore, the investigations of the epidermal-specific, inducible mouse models expressing individual cFLIP isoforms demonstrated that the presence of any of the isoforms was sufficient to rescue the phenotype of cFLIP deficiency from the characteristic weight loss and to ameliorate the macroscopic skin phenotype observed in epidermal cFLIP KO animals. However, histological analysis of skin samples have shown that the cFLIP S isoform is not sufficient to rescue the epidermal thickening and increased keratinocytes cell death. Moreover, in contrast to the typical apoptotic cell death in cFLIP KO keratinocytes, those expressing the cFLIP short isoform demonstrated a combination from both, caspase independent and caspase-dependent cell death. In summary, our data suggest that different cFLIP isoforms serve as a cell death switch regulating apoptosis/necroptosis decision in vivo and warrant future studies to dissect the impact of individual cFLIP isoforms in several inflammatory skin diseases, including psoriasis and toxic epidermal necrolysis (TEN).

Publications

• TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma. Cell Death & Differentiation, 26(12), 2520-2534.
  Podder, Biswajit; Guttà, Cristiano; Rožanc, Jan; Gerlach, Elke; Feoktistova, Maria; Panayotova-Dimitrova, Diana; Alexopoulos, Leonidas G.; Leverkus, Martin & Rehm, Markus
  
• A20 Promotes Ripoptosome Formation and TNF-Induced Apoptosis via cIAPs Regulation and NIK Stabilization in Keratinocytes. Cells, 9(2), 351.
  Feoktistova, Maria; Makarov, Roman; Brenji, Sihem; Schneider, Anne T.; Hooiveld, Guido J.; Luedde, Tom; Leverkus, Martin; Yazdi, Amir S. & Panayotova-Dimitrova, Diana
  
• TNF Is Partially Required for Cell-Death-Triggered Skin Inflammation upon Acute Loss of cFLIP. International Journal of Molecular Sciences, 21(22), 8859.
  Feoktistova, Maria; Makarov, Roman; Leverkus, Martin; Yazdi, Amir S. & Panayotova-Dimitrova, Diana
  
• RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation. International Journal of Molecular Sciences, 22(22), 12459.
  Feoktistova, Maria; Makarov, Roman; Yazdi, Amir S. & Panayotova-Dimitrova, Diana
  
• Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and KCa3.1 ion channels, but involves phosphoinositide 3-kinase. Pflügers Archiv -European Journal of Physiology, 475(3), 405-416.
  Cortés, Franco Klaus-Daniel; Brakmann, Ilka C.; Feoktistova, Maria; Panayotova-Dimitrova, Diana; Gründer, Stefan & Tian, Yuemin
  
• Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris. Vaccines, 10(2), 208.
  Schmidt, Morna F.; Feoktistova, Maria; Panayotova-Dimitrova, Diana; Eichkorn, Ramona A. & Yazdi, Amir S.
  
• Autolysosomal activation combined with lysosomal destabilization efficiently targets myeloid leukemia cells for cell death. Frontiers in Oncology, 13.
  Shah, Harshit; Stankov, Metodi; Panayotova-Dimitrova, Diana; Yazdi, Amir; Budida, Ramachandramouli; Klusmann, Jan-Henning & Behrens, Georg M. N.