Vangelis Kondylis

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood. Here we show that mice with IEC-specific knockout of FADD (FADDIEC-KO), an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis, prevented the development of spontaneous pathology in both the small intestine and colon of FADDIEC-KO mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis, prevented colitis development in FADDIEC-KO but not in NEMOIEC-KO mice, showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADDIEC-KO mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADDIEC-KO mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.

The Golgi apparatus: Lessons from Drosophila

Historically, Drosophila has been a model organism for studying molecular and developmental biology leading to many important discoveries in this field. More recently, the fruit fly has started to be used to address cell biology issues including studies of the secretory pathway, and more specifically on the functional integrity of the Golgi apparatus. A number of advances have been made that are reviewed below. Furthermore, with the development of RNAi technology, Drosophila tissue culture cells have been used to perform genome‐wide screens addressing similar issues. Last, the Golgi function has been involved in specific developmental processes, thus shedding new light on the functions of a number of Golgi proteins.

Drosophila Sec16 Mediates the Biogenesis of tER Sites Upstream of Sar1 through an Arginine-Rich Motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.

Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER

Ceramides are central intermediates of sphingolipid metabolism with critical functions in cell organization and survival. They are synthesized on the cytosolic surface of the endoplasmic reticulum (ER) and transported by ceramide transfer protein to the Golgi for conversion to sphingomyelin (SM) by SM synthase SMS1. In this study, we report the identification of an SMS1-related (SMSr) enzyme, which catalyses the synthesis of the SM analogue ceramide phosphoethanolamine (CPE) in the ER lumen. Strikingly, SMSr produces only trace amounts of CPE, i.e., 300-fold less than SMS1-derived SM. Nevertheless, blocking its catalytic activity causes a substantial rise in ER ceramide levels and a structural collapse of the early secretory pathway. We find that the latter phenotype is not caused by depletion of CPE but rather a consequence of ceramide accumulation in the ER. Our results establish SMSr as a key regulator of ceramide homeostasis that seems to operate as a sensor rather than a converter of ceramides in the ER.

ERK7 is a negative regulator of protein secretion in response to amino‐acid starvation by modulating Sec16 membrane association

RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic‐Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino‐acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C‐terminus of Sec16, a key ER exit site component. This post‐translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth.

NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis

Summary IκB kinase/nuclear factor κB (IKK/NF-κB) signaling exhibits important yet opposing functions in hepatocarcinogenesis. Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer. Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMOLPC-KO mice. Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMOLPC-KO mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1. Collectively, these results show that NEMO prevents hepatocarcinogenesis by inhibiting RIPK1 kinase activity-driven hepatocyte apoptosis through NF-κB-dependent and -independent functions.

Golgi ribbon unlinking: an organelle-based G2/M checkpoint.

Cell cycle checkpoints have mostly been associated with signaling mechanisms monitoring the genetic material for abnormalities and inaccuracy in partitioning, such as DNA lesions or failure in chromosome attachment to kinetochores. However, it becomes more evident that other checkpoints are turned on by cytoplasmic events, such as the cytoskeleton organisation and the organelle structure. Here, we summarize recent evidence strongly suggesting that the integrity of the Golgi ribbon, and more precisely the tubules interconnecting the Golgi stacks to form this ribbon, at late G2/early prophase, is linked to a Golgi-related G2/M checkpoint. A number of kinases phosphorylating a so far small subset of Golgi-localized proteins have been shown to promote the G2-specific Golgi ribbon unlinking allowing the cell to enter mitosis. When these kinases are inactivated or when the substrates cannot be phosphorylated, Golgi unlinking is prevented and the cells are blocked or delayed in G2 phase.

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

Summary Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Endosome-mediated autophagy: An unconventional MIIC-driven autophagic pathway operational in dendritic cells

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4BC74A mutant and atg4b−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.

The interplay of IKK, NF-κB and RIPK1 signaling in the regulation of cell death, tissue homeostasis and inflammation

Regulated cell death pathways have important functions in host defense and tissue homeostasis. Studies in genetic mouse models provided evidence that cell death could cause inflammation in different tissues. Inhibition of RIPK3‐MLKL‐dependent necroptosis by FADD and caspase‐8 was identified as a key mechanism preventing inflammation in epithelial barriers. Moreover, the interplay between IKK/NF‐κB and RIPK1 signaling was recognized as a critical determinant of tissue homeostasis and inflammation. NEMO was shown to regulate RIPK1 kinase activity‐mediated apoptosis by NF‐κB‐dependent and –independent functions, which are critical for averting chronic tissue injury and inflammation in the intestine and the liver. In addition, RIPK1 was shown to exhibit kinase activity‐independent functions that are essential for preventing cell death, maintaining tissue architecture and inhibiting inflammation. In the intestine, RIPK1 acts as a scaffold to prevent epithelial cell apoptosis and preserve tissue integrity. In the skin, RIPK1 functions via its RHIM to counteract ZBP1/DAI‐dependent activation of RIPK3‐MLKL‐dependent necroptosis and inflammation. Collectively, these studies provided evidence that the regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis, and suggested that cell death could be causally involved in the pathogenesis of inflammatory diseases.