ABSTRACT
Sphingolipids are an important part of the plasma membrane and implicated in a multitude of cellular processes. However, little is known about the role of sphingolipids in an epithelial context and their potential influence on the activity of signaling pathways. To shed light on these aspects we analyzed the consequences of changing ceramide levels in vivo in the Drosophila wing disc: an epithelial tissue in which the most fundamental signaling pathways, including the Wnt/Wg signaling pathway, are well characterized. We found that downregulation of Drosophila's only ceramide synthase gene schlank led to defects in the endosomal trafficking of proteins. One of the affected proteins is the Wnt ligand Wingless (Wg) that accumulated. Unexpectedly, although Wg protein levels were raised, signaling activity of the Wg pathway was impaired. Recent work has spotlighted the central role of the endocytic trafficking in the transduction of the Wnt signal. Our results underscore this and support the view that sphingolipid levels are crucial in orchestrating epithelial endocytic trafficking in vivo. They further demonstrate that ceramide/sphingolipid levels can affect Wnt signaling.
Subject(s)
Ceramides/deficiency , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Endosomes/metabolism , Oxidoreductases/metabolism , Wnt Proteins/metabolism , Wnt1 Protein/metabolism , Animals , Biological Transport , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation , Imaginal Discs/cytology , Imaginal Discs/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Sphingosine N-Acyltransferase , Transgenes , Wings, Animal/cytology , Wings, Animal/metabolism , Wnt Proteins/genetics , Wnt1 Protein/geneticsABSTRACT
Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.