Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway.

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.

The mammalian rhomboid protein RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets.

In metazoans, the architecture of the endoplasmic reticulum (ER) differs between cell types and undergoes major changes throughout the cell cycle and according to physiological needs. Although much is known about how the different ER morphologies are generated and maintained, especially ER tubules, how context-dependent changes in ER shape and distribution are regulated and the factors involved are less well characterized, as are the factors that contribute to the positioning of the ER within the cell. By overexpression and KO experiments, we show that the levels of RHBDL4, an ER-resident rhomboid protease, modulate the shape and distribution of the ER, especially during conditions that require rapid changes in the ER sheet distribution, such as ER stress. We demonstrate that RHBDL4 interacts with cytoskeleton-linking membrane protein 63 (CLIMP-63), a protein involved in ER sheet stabilization, as well as with the cytoskeleton. Furthermore, we found that mice lacking RHBDL4 are sensitive to ER stress and develop liver steatosis, a phenotype associated with unresolved ER stress. Taken together, these data suggest a new physiological role for RHBDL4 and also imply that this function does not require its enzymatic activity.

iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2.

The folding capacity of membrane and secretory proteins in the endoplasmic reticulum (ER) can be challenged by physiological and pathological perturbations, causing ER stress. If unresolved, this leads to cell death. We report a role for iRhom pseudoproteases in controlling apoptosis due to persistent ER stress. Loss of iRhoms causes cells to be resistant to ER stress-induced apoptosis. iRhom1 and iRhom2 interact with IP3 receptors, critical mediators of intracellular Ca2+ signalling, and regulate ER stress-induced transport of Ca2+ into mitochondria, a primary trigger of mitochondrial membrane depolarisation and cell death. iRhoms also bind to the anti-apoptotic regulator BCL-2, attenuating the inhibitory interaction between BCL-2 and IP3 receptors, which promotes ER Ca2+ release. The discovery of the participation of iRhoms in the control of ER stress-induced cell death further extends their potential pathological significance to include diseases dependent on protein misfolding and aggregation.

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration.

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here, we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.

A genome-wide association study in mice reveals a role for Rhbdf2 in skeletal homeostasis.

Low bone mass and an increased risk of fracture are predictors of osteoporosis. Individuals who share the same bone-mineral density (BMD) vary in their fracture risk, suggesting that microstructural architecture is an important determinant of skeletal strength. Here, we utilized the rich diversity of the Collaborative Cross mice to identify putative causal genes that contribute to the risk of fractures. Using microcomputed tomography, we examined key structural features that pertain to bone quality in the femoral cortical and trabecular compartments of male and female mice. We estimated the broad-sense heritability to be 50-60% for all examined traits, and we identified five quantitative trait loci (QTL) significantly associated with six traits. We refined each QTL by combining information inferred from the ancestry of the mice, ranging from RNA-Seq data and published literature to shortlist candidate genes. We found strong evidence for new candidate genes, particularly Rhbdf2, whose close association with the trabecular bone volume fraction and number was strongly suggested by our analyses. We confirmed our findings with mRNA expression assays of Rhbdf2 in extreme-phenotype mice, and by phenotyping bones of Rhbdf2 knockout mice. Our results indicate that Rhbdf2 plays a decisive role in bone mass accrual and microarchitecture.

Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation.

Macrophages are prominent cells in acute and chronic inflammatory diseases. Recent studies highlight a role for macrophage proliferation post-monocyte recruitment under inflammatory conditions. Using an acute peritonitis model, we identify a significant defect in macrophage proliferation in mice lacking the leukocyte transmembrane protease ADAM17. The defect is associated with decreased levels of macrophage colony-stimulating factor 1 (CSF-1) in the peritoneum and is rescued by intraperitoneal injection of CSF-1. Cell surface CSF-1 (csCSF-1) is one of the substrates of ADAM17. We demonstrate that both infiltrated neutrophils and macrophages are major sources of csCSF-1. Furthermore, acute shedding of csCSF-1 following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the neutrophil surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages and to prevent macrophage proliferation. In acute inflammation, csCSF-1 release and macrophage proliferation are self-limiting due to transient leukocyte recruitment and temporally restricted csCSF-1 expression. In chronic inflammation, such as atherosclerosis, the ADAM17-mediated lesional macrophage proliferative response is prolonged. Our results demonstrate a novel mechanism whereby ADAM17 promotes macrophage proliferation in states of acute and chronic inflammation.

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated.

The Tomato/GFP-FLP/FRT method for live imaging of mosaic adult Drosophila photoreceptor cells.

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

ER stress inhibits neuronal death by promoting autophagy.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.

ER stress protects from retinal degeneration.

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.