Identification of the molecular role of Pelota protein (PELO) by analysis of conditional Pelo-knockout mice
von Manar Mohamed Mansour El Kenani
Datum der mündl. Prüfung:2017-02-14
Betreuer:Prof. Dr. Ibrahim Adham
Gutachter:Dr. Laura C. Zelarayán
Gutachter:Prof. Dr. Martin Oppermann
EnglischThe skin acts as a first protective barrier that diminishes the fluid loss from the body and prevents the entry of toxic and pathogenic agents. Defect in the formation of the epidermal barrier results in neonatal lethality and increased susceptibility to skin inflammation. Here, we show that temporal depletion of Pelota (Pelo) during embryonic development disrupts the acquisition of epidermal barrier. The defective function of epidermal barrier in Pelo-deficient mice is a result of failed processing of profilaggrin into filaggrin monomers, which promote the formation of protective epidermal layer. Expression analysis revealed that the expression pattern of Pelo correlates with the epidermal barrier formation and resemble that of pSmad and pAKT. Further experiments showed that Pelo negatively regulates BMP-PI3K/AKT signaling pathways. Hence activation of BMP-PI3K/AKT was the underlying cause for the perturbed function of Pelo-deficient epidermis. Inhibition of PI3K/AKT and BMP signaling pathways by addition of PI3K inhibitor and Noggin respectively, in organotypic culture of mutant and control skin explants revealed that attenuated activity of PI3K/AKT did not significantly affect the BMP activity. In contrast, inhibition of BMP signaling resulted in a significant decrease in the PI3K/AKT activity in mutant skin and interestingly the restoration of the profilaggrin processing and the epidermal barrier function. In adult mice, deletion of Pelo resulted in epidermal hyperproliferation, abnormal differentiation of keratinocytes and cutaneous inflammation. These phenotypes are often associated with epidermal barrier dysfunction. To determine whether these symptoms are due to systemic immune-system disorder or a result of affected skin barrier, Pelo was topically deleted in a small region of the tail skin. Histological analyses revealed that hyperkeratosis and increased infiltration of mast cells were only restricted to the topically Pelo-deficient skin region and theother skin areas were completely normal. Additionally, our results showed the perturbation of the epidermal barrier function in Pelo-null skin explants grown in vitro providing strong evidence that the barrier defect was not due to systemic effect of Pelo deletion, but due to skin-autonomous effect. These results point out that Pelo is a novel modulator of the BMP-PI3K/AKT signaling axis which promotes the maturation of profilaggrin to ensure epidermal barrier formation. In the second part of the study, we investigated the molecular causes for attenuated rate of cell proliferation and impaired differentiation of Pelo-deficient ESCs. We found that the elevated activity of PI3K/AKT signaling is not the cause for impaired differentiation of Pelo-null ESCs. Despite the enhanced activity of PI3K/AKT in Pelo-null ESCs, Foxo1 escapes the inhibitory effect of PI3K/AKT signaling and retain in the nucleus. Nuclear Foxo1 tends to divert β-catenin from binding TCF and hereby restrain the WNT/TCF activity. Induction of ESC differentiation by retinoic acid (RA) led us to suggest that retinol metabolism in Pelo mutant ESCs is disrupted. As RDH is one targets of Foxo1, we assumed that the binding of Foxo1to β-catenin on one hand decreased the proliferative effect of Wnt signaling pathway and on the other hand altered the Foxo1 transcription activity. Therefore, the RDH transcription was affected resulting in defect in retinol metabolism to active retinoids, and probably this was the underlying cause for impaired differentiation of Pelo-mutant ESCs in absence of LIF. Extending the study on role of Pelo in ESCs self-renewal and differentiation would add valuable information about the role of PELO during early embryonic development, Moreover this knowledge may be used for improving somatic cell reprogramming as well.
Keywords: Pelota; epidermal permeability barrier; phosphoinositide 3-kinase; Bone morphogenetic protein; Embryonic stem cells